Compositions and methods for drug-sensitization or inhibition of a cancer cell

ABSTRACT

The disclosure provides rifamycin and rifamycin derivative compositions, including rifabutin and rifabutin derivative compositions able to cause drug-sensitization in a cancer cell or inhibition of a cancer cell. The disclosure also provides methods of administering such compositions to cancer cells to sensitize them to drugs, such as chemotherapeutics, or directly inhibit them. The disclosure also provides methods of administering such compositions to increase reactive oxygen species (ROS), particularly superoxides, in cancer cells. The disclosure further provides methods of determining whether a cancer will respond to chemotherapeutics and whether to administer rifamycin or a rifamycin derivative based on ROS levels in cancer cells of a patient.

CROSS-REFERENCE TO RELATED APPLICATION

The present application is continuation pending U.S. patent applicationSer. No. 14/322,727 filed Jul. 2, 2014; which is a continuation ofInternational Patent Application PCT/US2013/057369 filed Aug. 29, 2013;which claims priority under 35 U.S.C. §119 to U.S. Provisional PatentApplication Ser. No. 61/695,041 filed Aug. 30, 2012, and to U.S.Provisional Patent Application Ser. No. 61/784,416, filed Mar. 14, 2013.Each application is incorporated by reference herein in its entirety.

TECHNICAL FIELD

The present disclosure relates to compositions for drug-sensitization ofcancer cells. In particular, it relates to compositions includingrifamycin or a rifamycin derivative, such as rifabutin or a rifabutinderivative. The present disclosure also relates to methods ofsensitizing a cancer cell to another drug or combination of drugs byapplying rifamycin or a rifamycin derivative, such as rifabutin or arifabutin derivative to the cancer cell. The present disclosureadditionally relates to methods of damaging cancer cells by applyingrifamycin or a rifamycin derivative.

BACKGROUND

Cancer Therapeutics

Effective cancer treatment is frequently inhibited by the inability ofthe patient to withstand an effective dose of a therapeutic drug, by thedevelopment of resistance to therapeutic drugs by cancer cells, or both.These problems are exhibited across a wide range of cancers andtherapeutic drugs. Physicians and researchers have attempted to addressthese problems through various approaches, such as administeringmultiple therapeutic drugs at once or in series, but these solutions arenot optimal because they frequently pose additional risks to thepatient, such as increased rates of relapse, increased chances ofopportunistic infections due to increased length of treatment, andincreased chances of adverse drug reactions due to exposure to moredrugs.

Many of these problems could be avoided or lessened by rendering thecancer cells more sensitive to one or more therapeutic drugs. However,safe and effective methods for sensitizing cancer cells in such a mannerare lacking.

Rifamycin and Rifabutin

Rifabutin is a member of the rifamycin class of antibiotics. Rifabutinwas approved for use as an antibiotic in the United States in 1992.Although rifabutin has been tested for other antibiotic andanti-inflammatory uses, its most common use remains the treatment oftuberculosis and other Mycobacterium infections. Rifampicin, anothermember of the rifamycin class of antibiotics, was introduced in 1967 andis also used to treat tuberculosis and similar infections.

SUMMARY

The present disclosure, in one embodiment, relates to a compositionincluding a rifamycin derivative or a pharmaceutically acceptable salt,hydrate, or prodrug thereof. The derivative is operable to inducedrug-sensitization in a cancer cell. The derivative is also operable toinhibit a cancer cell.

According to another embodiment, the disclosure provides a method ofsensitizing a cancer cell to a drug by administering rifamycin or arifamycin derivative to the cancer cell in an amount and for a timesufficient to sensitize the cancer cell to the drug.

According to a third embodiment, the disclosure provides a method ofincreasing the amount of a chemotherapeutic in a cancer cell byadministering rifamycin or a rifamycin derivative in an amount and for atime sufficient to decrease activity of or inhibit a p-glycoprotein(P-gp) efflux pump in the cell by.

According to a fourth embodiment, the disclosure provides a method ofincreasing reactive oxygen species (ROS) in a cancer cell byadministering rifamycin or a rifamycin derivative to the cancer cell inan amount and for a time sufficient to increase ROS in the cancer cell.

According to a fifth embodiment, the disclosure provides a method ofinhibiting a cancer cell with a drug by administering rifamycin or arifamycin derivative to the cancer cell in an amount and for a timesufficient to sensitize the cancer cell to the drug and administeringthe drug to the cancer cell in an amount and for a time sufficient toinhibit the cancer cell. The amount or time of administration withrespect to the drug are less than that required to achieve the sameinhibition in the absence of rifamycin or a rifamycin derivative withrespect to a given cancer cell type.

A sixth embodiment of the disclosure relates to a method of increasingsusceptibility of a cancer cell to a drug by administering rifamycin ora rifamycin derivative to the cancer cell in an amount and for a timesufficient to increase reactive oxygen species (ROS) in the cancer celland administering the drug to the cancer cell in an amount and for atime sufficient to inhibit the cancer cell. The amount or time ofadministration with respect to the drug is less than that required toachieve the same inhibition in the absence of increased ROS.

A seventh embodiment of the disclosure provides a method of inhibiting acancer cell by administering rifamycin or a rifamycin derivative to thecancer cell in an amount and for a time sufficient to inhibit the cell.

According to an eighth embodiment, the disclosure provides a method ofincreasing susceptibility of a cancer cell to a drug by administeringrifamycin or a rifamycin derivative to a cancer cell in an amount andfor a time sufficient to increase the amount of the drug in the cancercell as compared to the amount of the drug that would be present in theabsence of the rifamycin or rifamycin derivative and administering thedrug to the cancer cell in an amount and for a time sufficient toinhibit the cancer cell.

According to a ninth embodiment, the disclosure provides a method ofincreasing susceptibility of a cancer cell to a drug by administeringrifamycin or a rifamycin derivative to the cancer cell in an amount andfor a time sufficient to inhibit a p-glycoprotein (P-gp) efflux pump inthe cell and administering the drug to the cancer cell in an amount andfor a time sufficient to inhibit the cancer cell, wherein the amount ortime are less than that required to achieve the same inhibition in theabsence of inhibition of the P-gp pump.

A tenth embodiment of the disclosure provides a method of determiningwhether to administer rifamycin or a rifamycin derivative to a patientwith cancer. The method includes obtaining a cancer cell sample from thepatient, measuring the reactive oxygen species (ROS) amount in thesample, and determining if the ROS amount is low for the cancer celltype. A low ROS level indicates that administration of rifamycin or arifamycin derivative to the patient may be beneficial

The following abbreviations are used throughout the specification:

-   CHOP—cyclophosphamide, doxorubicin, vincristine, prednisone-   NHL—non-Hodgkin's lymphoma-   ROS—reactive oxygen species-   RTI-x—designates a rifamycin derivative in which “x” is replaced by    an identification number used in the present specification to    designate a particular composition.-   DOX—doxorubicin.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawings will be provided by the Office upon request and paymentof the necessary fee.

A more complete understanding of the present embodiments and advantagesthereof may be acquired by referring to the following description takenin conjunction with the accompanying drawings, which depict embodimentsof the present disclosure, and in which like numbers refer to similarcomponents, and in which:

FIG. 1 illustrates a cellular network via which rifabutin or a rifabutinderivative may cause drug-sensitization and an exampledrug-sensitization effect in CHOP-resistant DLBCL cells;

FIG. 2A illustrates the effects of rifabutin on growth of CHOP-sensitive(CRL2631) NHL cells and CHOP-resistant (G3) NHL cells in the presence orabsence of CHOP as demonstrated by resazurin fluorescence;

FIG. 2B illustrates the effects of rifabutin on growth of CHOP-resistant(G3) NHL cells in the absence of CHOP (top panel) as compared to acontrol drug as demonstrated by resazurin fluorescence and in thepresence of varying dilutions of CHOP for 24 hrs (bottom panel);

FIG. 2C illustrates the effects of rifabutin on growth of anotherCHOP-resistant NHL cell line (SUDHL10-R) and the parental CHOP-sensitiveNHL cell line (SUDHL10-S) after 24 hrs (top panel) and 48 hrs (bottompanel) of treatment as demonstrated by resazurin fluorescence;

FIG. 3A illustrates the effects of rifabutin or rifabutin derivativesRTI-79 and RTI-176 on cell growth of primary human dermal fibroblastsboth with and without 2 uM Dox:

FIG. 3B illustrates the effects of doxorubicin and rifabutin on cellgrowth of primary human dermal fibroblasts;

FIG. 4 illustrates the effects of rifabutin on growth of CHOP-resistantlymphoma cells obtained by aspiration from a dog as demonstrated byresazurin fluorescence;

FIG. 5 illustrates the effects of rifabutin in combination with CHOP orCHOP alone on tumor burden in mm³ over time in SCID mice injected withCHOP-resistant (G3) NHL cells:

FIG. 6 illustrates the effects of CHOP or control solution with no CHOPon tumor burden in mm³ over time in SCID mice injected withCHOP-sensitive (CRL2631) NHL cells;

FIG. 7 illustrates the effects of reduced dosages of CHOP+rifabutin orcontrol solution with no CHOP or rifabutin on tumor burden in mm³ overtime in SCID mice injected with CHOP-resistant (G3) NHL cells;

FIG. 8 illustrates a Kaplan-Meier curve showing average life span forSCID mice injected with CHOP-resistant (G3) cells when treated witheither doxorubicin alone (DOX) or doxorubicin+rifabutin derivativeRTI-81 (DOX+NZ);

FIG. 9 illustrates the average tumor volume of chemo-resistant SK-OV-3xenografts in mice after control treatment with saline, treatment with3.3 mg/kg DOX, and treatment with 3.3 mg/kg DOX+25 mg/kg rifabutin overtime.

FIG. 10 illustrates the average tumor volume of multi-drug resistantcancer cell line (NCI/ADR-RES) xenografts in mice after controltreatment with saline, treatment with 7 mg/kg DOXIL® and treatment with7 mg/kg DOXIL®+25 mg/kg RTI-79 over time.

FIG. 11 illustrates the average tumor volume of multi-drug resistantcancer cell line (NCI/ADR-RES) xenografts in mice with multiple, largetumors after control treatment with saline, treatment with 7 mg/kgDOXIL®, and treatment with 7 mg/kg DOXIL®+25 mg/kg RTI-79 over time.

FIG. 12 illustrates the effects of rifabutin or RTI-79 on growth ofCHOP-resistant (G3) NHL cells;

FIG. 13 illustrates the effects of rifabutin or RTI-176 on growth ofCHOP-resistant (G3) NHL cells;

FIG. 14 illustrates the effects of rifabutin or RTI-81 on growth ofCHOP-resistant (G3) NHL cells;

FIG. 15 illustrates the interaction of rifabutin and doxorubicin onCHOP-sensitive (CRL2631) NHL cells;

FIG. 16 illustrates the interaction of RTI-79 and doxorubicin onCHOP-sensitive (CRL2631) NHL cells.

FIG. 17 illustrates the effects of rifabutin or RTI-82 onmultidrug-resistant breast cancer (MDA-MB-231) cells:

FIG. 18 illustrates the interaction of rifabutin with actinomycin D onmulti-drug resistant sarcoma (MES-SA-Dx5) cells;

FIG. 19 illustrates the interaction of rifabutin with menadione ondexamethasone resistant multiple myeloma (MM.1R) cells;

FIG. 20 illustrates the interaction of rifabutin and RTI-79 with andwithout doxorubicin at an 8:1 rifabutin or RTI-79:doxorubicin molarratio on multi-drug resistant cancer cell line (NCI/ADR-RES) cells:

FIG. 21 illustrates the interaction of RTI-79 and doxorubicin onmulti-drug resistant T lymphoblastoid leukemia (MOLT-4) cells;

FIG. 22 illustrates the effects of rifabutin and RTI-79 with and withoutdoxorubicin at an 8:1 rifabutin or RTI-79:doxorubicin molar ratio onovarian carcinoma (OVCAR8) cells;

FIG. 23 illustrates the effects of rifabutin and actinomycin D onmulti-drug resistant sarcoma (MES-SA-Dx5) cells.

FIG. 24 illustrates the effects of rifabutin and menadione ondexamethasone resistant multiple myeloma (MM.1R) cells;

FIG. 25 illustrates the interaction of rifabutin and mitoxantrone onosteosarcoma (U-2 OS) cells;

FIG. 26 illustrates the interaction of rifabutin with gemcitabine onmulti-drug resistant breast cancer (MDA-MB-231) cells;

FIG. 27 illustrates the interaction of rifabutin with paclitaxel onmyeloid leukemia cells (HL-60) cells;

FIG. 28 illustrates the interaction of rifabutin and camptothecin onovarian cancer (OVCAR-8) cells;

FIG. 29 illustrates the number of viable cells present after re-exposureto CHOP of CHOP-sensitive (CRL2631) cells to a full or half dose of CHOPin the presence or absence of rifabutin:

FIG. 30A illustrates a Western blot for phosphorylated Akt (pAkt) Akt,14-3-3ζ, and an actin control in CHOP-sensitive (CRL2631) andCHOP-resistant (G3) cells. FIG. 30B illustrates the effect of varyingamounts of Akt Inhibitor VIII on growth of G3 cells as demonstrated byresazurin fluorescence. FIG. 30C illustrates a Western blot forphosphorylated Akt (pAkt) Akt, 14-3-3ζ, and a Vimentin control in G3cells exposed or not exposed to Akt Inhibitor VIII;

FIG. 31 illustrates the amount of ROS in CHOP-sensitive (CRL2631) orCHOP-resistant (G3) cells before and after 101 ng/ml CHOP treatment(cyclophosphamide=240 ng/ml [0.83 uM]; Doxorubicin=33 ng/ml [0.057 uM];Vincristine=0.93 ng/ml [0.0045 uM]; Prednisone=67 ng/ml [0.828 uM].

FIG. 32 illustrates the ROS levels in distinct populations of cells inCHOP-sensitive (CRL2631) cells purified by flow cytometry;

FIG. 33 illustrates the number of viable cells present after treatmentof low-ROS CRL2631 cells and high-ROS CRL2631 cells with 101 ng/ml CHOPtreatment (cyclophosphamide=240 ng/ml [0.83 uM]; Doxorubicin=33 ng/ml[0.057 uM]; Vincristine=0.93 ng/ml [0.0045 uM]; Prednisone=67 ng/ml[0.828 uM];

FIG. 34 illustrates the effect on cell growth of varying amounts of CHOPin the presence or absence of 10 uM rifabutin on low-ROS CRL2631 cellsas demonstrated by resazurin fluorescence;

FIG. 35 illustrates the effect of 10 uM rifabutin on ROS inCHOP-resistant (G3) cells over time:

FIG. 36A provides a western-blot showing different ABCB1 protein levelsin si-ABCB1 and si-NC1 (control si-RNA) treated ADR-RES cells, as wellas the untreated ADR-RES cells and its parental drug-sensitive strainOVCAR8;

FIG. 36B shows the effects of rifabutin (RBT) on calcein-AM efflux inOVCAR8 than in ADR-RES cells.

FIG. 36C shows the effects of 5 μM rifabutin (RBT). RTI-79, and rifampin(RMP) on calcein-AM efflux and the further effects of ABCB1RNA-silencing;

FIG. 37A shows dose-response curves of various RTIs on calcein-AMefflux;

FIG. 37B shows the effects of various RTIs on 1 uM doxorubicin'stoxicity in G3 cells;

FIG. 37C shows the correlation between efflux inhibition effect and drugsensitizing ability for various RTI-x rifamycin derivatives;

FIG. 37D shows the comparison of doxorubicin fluorescence intensity inthe NCI/ADR-RES cells with rifabutin treatment or dimethyl sulfoxide(DMSO) control.

FIG. 38A shows the effects of MDR/P-gp inhibitors and two control drugs(carboxin, nifazoxinide) on ROS in doxorubicin-sensitive OVCAR8 cells;

FIG. 38B shows the effects of MDR/P-gp inhibitors and two control drugs(carboxin, nifazoxinide) on ROS in doxorubicin-resistant ADR-RES cells;

FIG. 38C shows the effects of MDR/P-gp inhibitors and two control drugs(carboxin, nifazoxinide) on ROS in doxorubicin-resistant G3 cells;

FIG. 39 shows staining of ADR-RES cells treated with RTI-79; ADR-REScells were infected 24 hrs with a baculovirus expressing a recombinantGFP protein fused with a mitochondrial localization signal (green);cells were stained with CellROX to detect ROS (red) or DAPI to detectnuclei (blue);

FIG. 40 shows the effects of cell-permeable calcium modulators (BAPTA,Verapamil) and a Complex 1 inhibitor (Rotenone) on ROS in G3 cells;

FIG. 41A shows the effects of P-gp inhibitors (Reserpine, Elacridar) onROS levels in ADR and OVCAR8 cells

FIG. 41B shows the effects of RTI-79 on ROS and calcium mobilization indoxorubicin-sensitive lymphoma (CRL2631, 10S, WSU) and ovarian carcinoma(OVCAR8) cells and doxorubicin-resistant lymphoma (G3R, 10R, WSUR)cells;

FIG. 41C shows the levels of ROS and calcium mobilization in moreCHOP-sensitive lymphoma (CRL2631, 10S, WSU) compared to the moreresistant derivative cell lines (G3, 10R, WSU-R), and in the moredoxorubicin-sensitive OVCAR8 versus the more resistant derivative cellline ADR.

FIG. 42 shows a time course of RTI-79 induction of ROS and calciummobilization in G3 cells; and

FIG. 43 shows the effects of siRNA knockdown of P-gp on induction of ROSand mobilization of calcium.

FIG. 44 shows the effects of rifabutin on G3 and CRL2631 cells in acollagen invasion 3D assay.

FIG. 45 shows the effects of rifabutin on G3 and CRL2631 cells in amodified Boyden chamber assay.

DETAILED DESCRIPTION

The present disclosure relates to compositions and methods fordrug-sensitization of a cancer cell or for inhibiting a cancer cell, aswell as methods for diagnosis of whether a cancer cell may respond to achemotherapeutic or to a composition described herein. Thesecompositions and methods are described in further detail below.

Unless otherwise indicated by the specific context of thisspecification, a cancer cell may include a cell of any type of cancer.Furthermore, it may include a cancer cell in a patient, either in acancerous growth, such as a tumor, or in isolation from other cancercells, such as during metastasis. The patient may be any animal. Inparticular, the patient may be a mammal, such as a human, a pet mammalsuch as a dog or cat, an agricultural mammal, such as a horse, cow, pig,sheep, or goat, or a zoo mammal. Although many embodiments herein areexpressed in terms of a cancer cell, the same or similar effects may beseen in groups of cancer cells in a patient.

Drug-sensitization, unless otherwise indicated by the specific contextof this specification, may include increased sensitivity to a drug,decreased resistance to a drug, or potentiation of a drug's activity orefficacy. Any effect may be measured using any methods accepted in theart. In a specific embodiment, drug-sensitization may be determined byan increased ability of the drug to inhibit a cell. Cellular inhibitionmay include killing the cell, such as via apoptisis or necrosis,reducing the growth of the cell, thus reducing the growth of the cancercontaining the cell, rendering the cell more susceptible to the immunesystem, preventing or reducing metastasis, reducing the size of a tumorcontaining the cell, or otherwise negatively affecting a cancer cell. Anincreased ability of the drug to inhibit a cancer cell may bedemonstrated by an ability to inhibit the cell with a reduced amount ofdrug or in a shorter period of time than in the absence ofdrug-sensitization. In the case of drug-resistant cancer cells, whichinclude cells with inherent or acquired resistance, drug-sensitizationmay result in a renewed or newly acquired ability of the drug to inhibita cancer cell or type of cancer cell.

Compositions

The present disclosure includes drug-sensitization compositions, such aschemosensitizer compositions, including rifamycin and rifamycinderivatives, such as rifabutin or rifabutin derivatives or rifampicinand rifampicin derivatives. The present disclosure also includescompositions for inhibition of cancer cells including rifamycin andrifamycin derivatives, such as rifabutin or rifabutin derivatives orrifampicin (also called rifampin) and rifampicin derivatives. Otherrifamycin derivates include rifapentine and rifalazil.

In certain embodiments, the present disclosure provides derivatives ofrifabutin according to one of the following general structures:

in which R may be an alkyl, aryl, or hetero aryl group.

In other embodiments, the present disclosure provides enantiomers of thegeneral structures. In particular embodiments, it provides enantiomerswith the following general chrial structures:

in which R may be an alkyl, aryl, or hetero aryl group.

In certain embodiments having general structures I or II or generalchiral structures Ia or IIa, R may be one of the following structures:

In certain embodiments, the present disclosure provides derivatives ofrifabutin according to the following formula:

In certain embodiments, the present disclosure provides derivatives ofrifabutin according to the following formula:

In certain embodiments, the present disclosure provides derivatives ofrifabutin according to the following formula:

where X and R may include the following combinations:

The structure with the general formula above may also be the followingenantiomer:

In certain embodiments, the present disclosure provides derivatives ofrifabutin according to the following formula:

In certain embodiments having general structures III or IV or generalchiral structures IIIa or IVa, R may be one of the following structures:

wherein X is a C, O, or N and R is an alkyl, aryl, or hetero-aryl group.

In another embodiment, the present disclosure provides derivatives ofrifabutin according to the following formula:

wherein X is a C, O, or N and R is an alkyl, aryl, or hetero-aryl groupor wherein X and R are as follows:

In one embodiment, a composition of the general formula above may be thefollowing enantiomer

In certain embodiments, the present disclosure provides derivatives ofrifabutin according to the following formula:

wherein X is a C, O, or N and R may include the structures listed below:

In certain embodiments, the present disclosure provides derivatives ofrifabutin according to the following formula, wherein X is a C, O, or N:

In certain embodiments, a composition with the general formula above maybe the following enantiomer:

In certain embodiments, the present disclosure provides derivatives ofrifabutin according to the following formula:

In certain embodiments, the present disclosure provides derivatives ofrifabutin according to the following formula:

wherein X is a C, O, or N and R is an alkyl, aryl, or hetero-aryl groupor wherein X and R are as follows:

In certain embodiments, the present disclosure provides derivatives ofrifabutin according to the following formula:

In other embodiments, the present disclosure provides adrug-sensitization composition including a series of 3,4-cyclo-rifamycinderivatives. Examples of such compositions are as follows:

or the following enantiomer:

In certain embodiments X may be CH, S, SO, SO₂ or N. Y may be H or anacetyl group. R1 may be hydrogen. R2 may be a hydroxyl or an amino(—NH₂) group. R1 and R2 together may be an oxo or imine group. R3 may beone of the following groups: hydrogen, alkyl, alkenyl, alkynyl,cycloalkyl, aryl, heteroaryl and heterocycloalkyl groups that may beadditionally substituted with from zero to four substituents chosenindependently from halogen, hydroxy, alkoxy-alkyl, —CN, nitro, —S-alkyl,amino, alkylamino, dialkylamino, dialkylaminoalkyl, carboxy,carboalkoxy, acyl, carboxamido, alkylsulfoxide, acylamino, phenyl,benzyl, phenoxy, and benzyloxy. In certain embodiments. R3 may be—C(═O)—R4, —C(═O)—O—R4 and —C(═O)—NH—R4 where R4 is independentlyselected from alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl andheterocycloalkyl groups that may be additionally substituted with fromzero to four substituents chosen independently from halogen, hydroxy,alkoxy-alkyl, —CN, nitro, —S-alkyl, amino, alkylamino, dialkylamino,dialkylaminoalkyl, carboxy, carboalkoxy, acyl, carboxamido,alkylsulfoxide, acylamino, phenyl, benzyl, phenoxy and benzyloxy.

In other embodiments, the present invention provides compositions of thefollowing structure:

or the following enantiomer:

wherein Y is H or an acetyl group and R4 may be selected from alkyl,alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl and heterocycloalkylgroups that may be additionally substituted with from zero to foursubstituents chosen independently from halogen, hydroxy, alkoxy-alkyl,—CN, nitro, —S-alkyl, amino, alkylamino, dialkylamino,dialkylaminoalkyl, carboxy, carboalkoxy, acyl, carboxamido,alkylsulfoxide, acylamino, phenyl, benzyl, phenoxy and benzyloxy.

In certain embodiments, the present invention provides compositions withthe following structure:

or the following enantiomer:

wherein Y is H, or acetyl group; Z is carbon, oxygen or nitrogen atom;and R4 is independently selected from alkyl, alkenyl, alkynyl,cycloalkyl, aryl, heteroaryl and heterocycloalkyl groups that may beadditionally substituted with from zero to four substituents chosenindependently from halogen, hydroxy, alkoxy-alkyl, —CN, nitro, —S—alkyl, amino, alkylamino, dialkylamino, dialkylaminoalkyl, carboxy,carboalkoxy, acyl, carboxamido, alkylsulfoxide, acylamino, phenyl,benzyl, phenoxy and benzyloxy.

Examples of drug-sensitization compositions in accordance with certainaspects of the present disclosure may include those listed in Table 1.Compositions of Table 1 are designated by like names throughout thisspecification.

TABLE 1 Rifamycin Derivatives RTI- General x structure R Name  33 I

11-deoxy-11-imino-4- deoxy-3,4[2-spiro-[1-(t- butyloxycarbonyl)-piperidin-4-yl]]-(1H)- imidazo-(2,5- dihydro)rifamycin S  44 I —H11-deoxy-11-imino-4- deoxy-3,4[2-spiro- [piperidin-4-yl]]-(1H)-imidazo-(2,5- dihydro)rifamycinS  49 I

11-deoxy-11-imino-4- deoxy-3,4[2-spiro-[1- (benzyl)-piperidin-4-yl]]-(1H)-imidazo-(2,5- dihydro)rifamycin S  51 I

11-deoxy-11-imino-4- deoxy-3,4[2-spiro-[1-(2- methoxyethyl)-piperidin-4-yl]]-(1H)- imidazo-(2,5- dihydro)rifamycin S  53 I

11-deoxy-11-imino-4- deoxy-3,4[2-spiro-[1-(2- morpholinoethyl)-piperidin-4-yl]]-(1H)- imidazo-(2,5- dihydro)rifamycin S  57 I

11-deoxy-11-imino-4- deoxy-3,4[2-spiro-[1- (cyclobutylmethyl)-piperidin-4-yl]]-(1H)- imidazo-(2,5- dihydro)rifamycin S  59 I

11-deoxy-11-imino-4- deoxy-3,4[2-spiro-[1- (cyclopropylmethyl)-piperidin-4-yl]]-(1H)- imidazo-(2,5- dihydro)rifamycin S  60 I

11-deoxy-11-imino-4- deoxy-3,4[2-spiro-[1- (isopropyl)-piperidin-4-yl]]-(1H)- imidazo-(2,5- dihydro)rifamycin S  61 I

11-deoxy-11-imino-4- deoxy-3,4[2-spiro-[1-(t- ethyloxycarbonyl)-piperidin-4-yl]]-(1H)- imidazo-(2,5- dihydro)rifamycin S  63 I

11-deoxy-11-imino-4- deoxy-3,4[2-spiro-[1- (acetyl)-piperidin-4-yl]]-(1H)-imidazo-(2,5- dihydro)rifamycin S  64 I

11-deoxy-11-imino-4- deoxy-3,4[2-spiro-[1-(n- propyl)-piperidin-4-yl]]-(1H)-imidazo-(2,5- dihydro)rifamycin S  65 I

11-deoxy-11-imino-4- deoxy-3,4[2-spiro-[1- (cyclopropyl)-piperidin-4-yl]]-(1H)-imidazo-(2,5- dihydro)rifamycin S  66 I

11-deoxy-11-imino-4- deoxy-3,4[2-spiro-[1- (ethyl)-piperidin-4-yl]]-(1H)-imidazo-(2,5- dihydro)rifamycin S  67 I

11-deoxy-11-imino-4- deoxy-3,4[2-spiro-[1- (beRTIoyl)-piperidin-4-yl]]-(1H)-imidazo-(2,5- dihydro)rifamycin S  68 I

11-deoxy-11-imino-4- deoxy-3,4[2-spiro-[1- (benzyloxycarbonyl)-piperidin-4-yl]]-(1H)- imidazo-(2,5- dihydro)rifamycin S  69 I —CH₃11-deoxy-11-imino-4- deoxy-3,4[2-spiro-[1- (methyl)-piperidin-4-yl]]-(1H)-imidazo-(2,5- dihydro)rifamycin S  70 I

11-deoxy-11-imino-4- deoxy-3,4[2-spiro-[1-(2- methylpropyl)-piperidin-4-yl]]-(1H)-imidazo-(2,5- dihydro)rifamycin S  74 I

11-deoxy-11-imino-4- deoxy-3,4[2-spiro-[1- (phenylaminocarbonyl)-piperidin-4-yl]]-(1H)- imidazo-(2,5- dihydro)rifamycin S  75 II

4-deoxy-3,4[2-spiro-[1- (t-butyloxycarbonyl)- piperidin-4-yl]]-(1H)-imidazo-(2,5- dihydro)rifamycin S  76 II

4-deoxy-3,4[2-spiro-[1- (ethyloxycarbonyl)- piperidin-4-yl]]-(1H)-imidazo-(2,5- dihydro)rifamycin S  77 I

11-deoxy-11-imino-4- deoxy-3,4[2-spiro-[1- (ethyloxycarbonyl-piperidin-4-yl]]-(1H)- imidazo-(2,5- dihydro)rifamycin S  78 II

4-deoxy-3,4[2-spiro-[1- (n-propyloxycarbonyl)- piperidin-4-yl]]-(1H)-imidazo-(2,5- dihydro)rifamycin S  79 II

4-deoxy-3,4[2-spiro-[1- (isobutyloxycarbonyl)- piperidin-4-yl]]-(1H)-imidazo-(2,5- dihydro)rifamycin S  80 II

4-deoxy-3,4[2-spiro-[1- (benzyloxycarbonyl)- piperidin-4-yl]]-(1H)-imidazo-(2,5- dihydro)rifamycin S  81 I

11-deoxy-11-imino-4- deoxy-3,4[2-spiro-[1- (isobutyloxycarbonyl)-piperidin-4-yl]]-(1H)- imidazo-(2,5- dihydro)rifamycin S  82 I

11-deoxy-11-imino-4- deoxy-3,4[2-spiro-[1- (ethylaminocarbonyl)-piperidin-4-yl]]-(1H)- (imidazo-(2,5- dihydro)rifamycin S  83 II

4-deoxy-3,4[2-spiro-[1- (ethylaminocarbonyl)- piperidin-4-yl]]-(1H)-imidazo-(2,5- dihydro)rifamycin S  84 I

11-deoxy-11-imino-4- deoxy-3,4[2-spiro-[1- (isopropyloxycalbonyl)-piperidin-4-yl]]-(1H)- imidazo-(2,5- dihydro)rifamycin S  85 II

4-deoxy-3,4[2-spiro-[1- (isopropyloxycarbonyl)- piperidin-4-yl]]-(1H)-imidazo-(2,5- dihydro)rifamycin S  86 II

4-deoxy-3,4[2-spiro-[1- (phenylaminocarbonyl)- piperidin-4-yl]]-(1H)-imidazo-(2,5- dihydro)rifamycin S  87 II

4-deoxy-3,4[2-spiro-[1- (acetyl)-piperidin-4-yl]]- (1H)-imidazo-(2,5-dihydro)rifamycin S  88 II

4-deoxy-3,4[2-spiro-[1- (beRTIoyl)-piperidin-4- yl]]-(1H)-imidazo-(2,5-dihydro)rifamycin S  89 II

4-deoxy-3,4[2-spiro-[1- (3,3-dimethylbutanoyl)- piperidin-4-yl]]-(1H)-imidazo-(2,5- dihydro)rifamycin S  91 I

11-deoxy-11-imino-4- deoxy-3,4[2-spiro-[1- (3,3-dimethylbutanoyl)-piperidin-4-yl]]-(1H)- imidazo-(2,5- dihydro)rifamycin S  94 I

11-deoxy-11-imino-4- deoxy-3,4[2-spiro-[1-(n- pentanoyl)-piperidin-4-yl]]-(1H)-imidazo-(2,5- dihydro)rifamycin S  97 I

11-deoxy-11-imino-4- deoxy-3,4[2-spiro-[1-(2- methylpropanoyl)-piperidin-4-yl]]-(1H)- imidazo-(2,5- dihydro)rifamycin S  98 I

11-deoxy-11-imino-4- deoxy-3,4[2-spiro-[1- (3-methylbutanoyl)-piperidin-4-yl]]-(1H)- imidazo-(2,5- dihydro)rifamycin S 101 II

4-deoxy-3,4[2-spiro-[1- (dimethylaminocarbonyl)- piperidin-4-yl]]-(1H)-imidazo-(2,5- dihydro)rifamycin S 102 II

4-deoxy-3,4[2-spiro-[1- (isobutylaminocarbonyl)- piperidin-4-yl]]-(1H)-imidazo-(2,5- dihydro)rifamycin S 103 II

4-deoxy-3,4[2-spiro-[1- (isopropylaminocarbonyl)- piperidin-4-yl]]-(1H)-imidazo-(2,5- dihydro)rifamycin S 104 II

4-deoxy-3,4[2-spiro-[1- ((1-methylpropyl) aminocarbonyl)-piperidin-4-yl]]-(1H)- imidazo-(2,5- dihydro)rifamycin S 105 II

4-deoxy-3,4[2-spiro-[1- (t-butylaminocarbonyl)- piperidin-4-yl]]-(1H)-imidazo-(2,5- dihydro)rifamycin S 174 IV

11-deoxy-11-hydroxy-4- deoxy-3,4[2-spiro-[1-(2- methylpropyl)-piperidin-4-yl]]-(1H)-imidazo-(2,5- dihydro)rifamycin S 175 IV

11-deoxy-11-hydroxy-4- deoxy-3,4[2-spiro-[1- (isobutyloxycarbonyl)-piperidin-4-yl]]-(1H)- imidazo-(2,5- dihydro)rifamycin S 176 III

11-deoxy-11-amino-4- deoxy-3,4[2-spiro-[1- (isobutyloxycarbonyl)-piperidin-4-yl]]-(1H)- imidazo-(2,5- dihydro)rifamycin S 181 III

11-deoxy-11-amino-4- deoxy-3,4[2-spiro-[1-(2- methylpropyl)-piperidin-4-yl]]-(1H)- imidazo-(2,5- dihydro)rifamycin S 182 I

11-deoxy-11-imino-4- deoxy-3,4[2-spiro-[1- (isobutylaminocarbonyl)-piperidin-4-yl]]-(1H)- imidazo-(2,5- dihydro)rifamycin S 183 III

11-deoxy-11-amino-4- deoxy-3,4[2-spiro-[1- (isobutylaminocarbonyl)-piperidin-4-yl]]-(1H)- imidazo-(2,5- dihydro)rifamycin S 197 V

11-deoxy-11-hydroxyimino-4-deoxy- 3,4[2-spiro-[1- (isobutyloxycarbonyl)-piperidin-4-yl]]-(1H)- imidazo-(2,5- dihydro)rifamycin S 217 V

11-deoxy-11- hydroxyimino-4-deoxy- 3,4[2-spiro-[1-(isobutylaminocarbonyl)- piperidin-4-yl]]-(1H)- imidazo-(2,5-dihydro)rifamycin S

Modification of the rifamycin structure in locations corresponding tothe 21-OH, 23-OH or 25-O-Ac sites of the rifabutin structures I, II,III, IV and V do not generally affect drug-sensitization activity andthus variations with modifications at these sites or even elimination ofthese sites are encompassed herein. Such variations may be used toimprove synthesis yields, control costs, increase water solubility, orimprove pharmaceutical properties of the composition. Sites 21, 23 and25 are located as follows:

The present disclosure also includes pharmaceutically acceptable salts,hydrates, prodrugs, and mixtures of any of the above compositions. Theterm “pharmaceutically acceptable salt” refers to salts whose counterion derives from pharmaceutically acceptable non-toxic acids and bases.

The 3,4-cyclo-rifamycin derivatives which contain a basic moiety, suchas, but not limited to an amine or a pyridine or imidazole ring, mayform salts with a variety of organic and inorganic acids. Suitablepharmaceutically acceptable (i.e., non-toxic, physiologicallyacceptable) base addition salts for the compounds of the presentinvention include inorganic acids and organic acids. Examples includeacetate, adipate, alginates, ascorbates, aspartates, benzenesulfonate(besylate), benzoate, bicarbonate, bisulfate, borates, butyrates,carbonate, camphorsulfonate, citrate, digluconates, dodecylsulfates,ethanesulfonate, fumarate, gluconate, glutamate, glycerophosphates,hemisulfates, heptanoates, hexanoates, hydrobromides, hydrochloride,hydroiodides, 2-hydroxyethanesulfonates, isethionate, lactate, maleate,malate, mandelate, methanesulfonate, 2-naphthalenesulfonates,nicotinates, mucate, nitrate, oxalates, pectinates, persulfates,3-phenylpropionates, picrates, pivalates, propionates, pamoate,pantothenate, phosphate, salicylates, succinate, sulfate, sulfonates,tartrate, p-toluenesulfonate, and the like.

The 3,4-cyclo-rifamycin derivatives which contain an acidic moiety, suchas, but not limited to a carboxylic acid, may form salts with variety oforganic and inorganic bases. Suitable pharmaceutically acceptable baseaddition salts for the compounds of the present invention include, butare not limited to, ammonium salts, metallic salts made from calcium,lithium, magnesium, potassium, sodium and zinc or organic salts madefrom lysine, N,N-dialkyl amino acid derivatives (e.g.N,N-dimethylglycine, piperidine-1-acetic acid and morpholine-4-aceticacid), N,N′-dibenzylethylenediamine, chloroprocaine, choline,diethanolamine, ethylenediamine, meglumine (N-methylglucamine),t-butylamine, dicyclohexylamine, hydrabamine, and procaine.

The 3,4-cyclo-rifamycin derivatives, and salts thereof, may exist intheir tautomeric form (for example, as an amide or imino ether). Allsuch tautomeric forms are contemplated herein as part of the presentinvention.

The compounds described herein may contain asymmetric centers and maythus give rise to enantiomers, diastereomers, and other stereoisomericforms. Each chiral center may be defined, in terms of absolutestereochemistry, as (R)- or (S)-. The present invention is meant toinclude all such possible isomers, as well as, their racemic andoptically pure forms. Optically active (R)- and (S)-, or (D)- and(L)-isomers may be prepared using chiral synthons or chiral reagents, orresolved using conventional techniques. When the compounds describedherein contain olefinic double bonds or other centers of geometricasymmetry, and unless specified otherwise, it is intended that thecompounds include both E and Z geometric isomers.

The configuration of any carbon-carbon double bond appearing herein isselected for convenience only and unless explicitly stated, is notintended to designate a particular configuration. Thus the carbon-carbondouble bond depicted arbitrarily above as E may be Z. E, or a mixture ofthe two in any proportion.

Abbreviations as used herein have the meanings known by one skilled inthe art. Specifically, Ac represent acetyl group, Boc representst-butoxycarbonyl group, Bn represents benzyl group, DCM representsdichloromethane, DMF represents N,N-dimethylformamide, DMSO representsdimethyl sulfoxide, Et represents ethyl group, EtOAc representsethylacetate, Me represents methyl group, Ph represents phenyl group,TEA represents triethylamine, TFA represents trifluoroacetic acid. THFrepresents tetrahydrofuran, and TMS is trimethylsilane group.

Compositions of the present disclosure may also include apharmaceutically acceptable carrier, in particular a carrier suitablefor the intended mode of administration, or salts, buffers, orpreservatives. Rifamycin and many of its derivatives, such as rifabutinand rifabutin derivatives are poorly soluble in water. Accordingly,aqueous compositions of the present disclosure may include solubilityenhancers. Compositions for oral use may include components to enhanceintestinal absorption. The overall formulation of the compositions maybe based on the intended mode of administration. For instance, thecomposition may be formulated as a pill or capsule for oral ingestion.In other examples, the composition may be encapsulated, such as in aliposome or nanoparticle. In particular, it may be encapsulated with thedrug to sensitize the cancer cell, such as encapsulated in a liposomewith doxorubicin. It may also be administered with a liposomal ornanoparticle drug, such as DOXIL® (doxorubicin HCl liposome injection)(Centocor Ortho Biotech Products, LP, Raritan, N.J.), whetherencapsulated with the drug or not. It may also be separatelyencapulsated.

Compositions of the present disclosure may contain a sufficient amountof rifamycin or rifamycin derivative to cause drug-sensitization orother inhibition of a cancer cell to occur when the composition isadministered to a cancer cell. The amount of rifamycin or rifamycinderivative, such as rifabutin or rifabutin derivative may vary dependingon other components of the composition and their effects on drugavailability in a patient, the type of drug or drugs to which the cancercell is sensitized, the amount of drug otherwise required to inhibit thecancer cell, the intended mode of administration, the intended schedulefor administration, any drug toxicity concerns, drug-drug interactions,such as interactions with other medications used by the patient, or theindividual response of a patient. Many compositions may contain anamount of rifamycin or rifamycin derivative, such as rifabutin orrifabutin derivative, well below levels at which toxicity to normalcells or to the patient overall becomes a concern.

Compositions of the present disclosure may also contain one or moredrugs for which the rifamycin or rifamycin derivative, such as rifabutinor rifabutin derivative, causes drug-sensitization. Example drugs aredescribed in the current specification. In another embodiment,compositions of the present disclosure may contain one or more otherdrugs commonly used in combination with the drug for which sensitizationoccurs. For example, a composition may include rifabutin or a rifabutinderivative with any CHOP drug, regardless of whether rifabutin causesdrug-sensitization for that drug. In still another embodiment, thecomposition may contain another drug that also causes drugsensitization, such as a drug that affects the amount or ROS,particularly superoxide, in a cell. For example it may containsuperoxide dismutase inhibitors. In still another embodiment, thecomposition may contain another drug that affects drug resistance or aproperty causing drug resistance in cancer cells. For example, it maycontain drugs affecting the apoptotic pathway, such as the apoptoticpathway inhibitors for Bcl-XL or mimetics for BH3 proteins.

Compositions of the present disclosure may further include othertherapeutic agents. For example, they may include any one or more of thechemotherapeutic agents listed herein, particularly those describedbelow in connection with Drug Sensitization Methods. The amounts ofthose chemotherapeutic agents in compositions of the present disclosuremay be reduced as compared to normal doses of such agents administeredin a similar fashion.

The amount of rifamycin or rifamycin derivative, such as rifabutin or arifabutin derivative, present in a composition may be measured in any ofa number of ways. The amount may, for example, express concentration ortotal amount. Concentration may be for example, weight/weight,weight/volume, moles/weight, or moles/volume. Total amount may be totalweight, total volume, or total moles. Typically, the amount of rifamycinor rifamycin derivative may be expressed in a manner standard for thetype of formulation or dosing regimen used.

The present disclosure further includes methods of identifying whether arifamycin derivative, such as a rifabutin derivative is able tosensitize a cancer cell or inhibit a cancer cell. Such methods includepreparing or obtaining such a derivative, applying it to a cancer cell,and identifying that the derivative renders the cancer cell moresusceptible to a chemotherapeutic in any manner described herein.

Drug-Sensitization Methods

The present disclosure also includes drug-sensitization methods in whicha rifamycin or rifamycin derivative, such as rifabutin or rifabutinderivative, composition is administered to a cancer cell in order tosensitize the cancer cell to another drug. The composition may be anycomposition described above. In a specific embodiment, the compositionmay be administered with any other drug which may alternatively bepresent in a pharmaceutical composition as described herein. For examplethe other drug may include DOXIL®.

The drug may be any drug for which rifamycin or a rifamycin derivative,such as rifabutin or a rifabutin derivative, increasesdrug-sensitization. In a specific embodiment, the drug may be achemotherapeutic. Example types of chemotherapeutics include alkylatingagents, antimetabolites, anti-tumor antibiotics, hormonal agents,targeted therapies, differentiating agents and other drugs.

Example alkylating agents include nitrogen mustards such asmechlorethamine (nitrogen mustard), chlorambucil, cyclophosphamide,ifosfamide, and melphalen. Example alkylating agents further includenitrosoureas, such as streptozocin, carmustine (BCNU), and lomustine.Example alkylating agents further include alkyl sulfonates such asbusulfan, triazines, such as procarbazine and dacarbazine (DTIC) andtemozolomide, and ethylenimines, such as thiotepa and altretamine(hexamethylmelamine). Example alkylating agents further include platinumdrugs, such as cisplatin, carboplatin, and oxaliplatin.

Example antimetabolites include purine antagonists such asmercaptopurine (6-MP), thioguanine (6-TG), fludarabine phosphate,clofarabine, cladribine, and pentostatin. Example antimetabolites alsoinclude pyrimidine antagonists such as fluorouracil (5-FU), floxuridine,capecitabine, cytarabine, gemcitabine and azacitidine. Exampleantimetabolites further include plant alkaloids. Some plant alkaloidsinclude topoisomerase inhibitors such as topoisomerase I inhibitors suchas camptothecin, topotecan and irinotecan, or topoisomerase IIinhibitors such as amsacrine, etoposide, and teniposide. Other plantalkyloids include mitotic inhibitors such as taxanes, includingpaclitaxel and docetaxel, epothilones, including ixabcpilone, vincaalkaloids, including vinblastine, vincristine, and vinorelbine, as wellas estramustine. Example antimetabolites further include folateantimetabolites such as methotrexate and pemetrexed. Otherantimetabolites include hydroxyurea.

Example anti-tumor antibiotics include anthracyclines or anthracyclineanalogs such as daunorubicin, doxorubicin, epirubicin, mitoxantrone, andidarubicin. Other anti-tumor antibiotics include dactinomycin,plicamycin, mitomycin, bleomycin, apicidin, and actinomycin.

Example hormonal agents include gonadotropin-releasing hormone agonistssuch as leuprolide and goserelin. Other example hormonal agents includearomatase inhibitors such as aminoglutethimide, exemestane, letrozoleand anastrozole. Other hormonal agents include tamoxifen and flutamide.Still other example hormonal agents include anti-estrogens such asfulvestrant, tamoxifen, and toremifene or anti-androgens such asbicalutamide, flutamide, and nilutamde. Example hormonal agents furtherinclude progestins such as megestrol acetate, and estrogens.

Example targeted therapies include antibodies or other therapeutics thatact on a molecular level such as imatinib, gefitinib, sunitinib, andbortezomib.

Example differentiating agents include retinoids such as tretinoin,bexarotene, and arsenic trioxide.

Other chemotherapeutics include L-asparaginase, phenoxodiol, rapamycin,and menadione.

In methods of the current disclosure, the cancer cell may be sensitizedto a drug already known to inhibit the cancer cell, or it may besensitized to a drug not previously used with that type of cancer cell.If the cancer cell is a drug-resistant cancer cell that has acquiredresistance, it may be sensitized to a drug that previously exhibited adecreased ability to inhibit the cancer cell or cancer cells of the sametype.

In another embodiment, the composition may directly inhibit the cancercell instead of or in addition to causing drug-sensitization.

The cancer cell that undergoes drug-sensitization or inhibition may beany type of cancer cell. It may, for instance, be a carcinoma, asarcoma, a leukemia, a lymphoma, or a glioma. It may also be a softcancer or a hard cancer. It may also be a cancer affecting a particularorgan or tissue, such as: an immunological-related cancer such asleukemia, lymphoma, including Non-Hodgkin's lymphoma, or Hodgkin'sdisease, myeloma, including multiple myeloma, sarcoma, lung cancer,breast cancer, ovarian cancer, uterine cancer, including endometrialcancer, testicular cancer, intestinal cancer, including colon cancer,rectal cancer, and small intestinal cancers, stomach cancer, esophagealcancer, oral cancer, pancreatic cancer, liver cancer, prostate cancer,glandular cancers such as adrenal gland cancer and pituitary tumor, bonecancer, bladder cancer, brain and other nervous tissue cancers,including glioma, eye cancer, including retinoblastoma, skin cancer,including basal cell carcinoma and melanoma, and kidney cancer.

The composition may be delivered to the cancer cell in a patient bydelivering the composition to the patient. The mode of delivery may beselected based on a number of factors, including metabolism of therifamycin or rifamycin derivative, such as the rifabutin or rifabutinderivative, or another drug in the composition, mode of administrationof other drugs to the patient, such as the drug to which the cancer cellis sensitized, the location and type of cancer cell to bedrug-sensitized, health of the patient, ability or inability to useparticular dosing forms or schedules with the patient, preferred dosingschedule, including any adjustment to dosing schedules due to sideeffects of chemotherapeutics, and ease of administration. In specificembodiments, the mode of administration may be enteral, such as orallyor by introduction into a feeding tube. In other specific embodiments,the mode of administration may be parenteral, such as intravenously.

The dosage amounts of the rifamycin or rifamycin derivative, such asrifabutin or rifabutin derivative and administration schedule may varydepending on other components of the composition and their effects ondrug availability in a patient, the type of drug or drugs to which thecancer cell is sensitized, the intended mode of administration, theintended schedule for administration, when other drugs are administered,any drug toxicity concerns, and the patient's response to the drug. In aspecific embodiment, the amount and frequency of rifamycin or rifamycinderivative such as rifabutin or rifabutin derivative delivered may besuch that levels in the patient remain well below levels at whichtoxicity to normal cells or to the patient becomes a concern. Howeverthe amount and frequency may also be such that the rifamycin orrifamycin derivative, such as rifabutin and rifabutin derivative, levelsin the cancer cell remain continuously at a level sufficient to inducedrug-sensitization or are at a level sufficient to induce-drugsensitization when or shortly after the drug to which the cancer cell issensitized is delivered to it. Accordingly, the rifabutin or rifabutinderivative composition may be taken on a regular basis during treatmentwith the drug to which the cancer cell is sensitized or it may be takenonly a set time before, at the same time, or a set time after the drugto which the cancer cell is sensitized.

Cancer Inhibition Methods

In some specific embodiments, the disclosure provides methods ofinhibiting a cancer cell using a drug to which the cancer cell isresistant by administering rifamycin or a rifamycin derivative, such asrifabutin or a rifabutin derivative, to the cancer cell.

In other specific embodiments, the disclosure provides methods ofreducing the amount of a drug administered to a patient by alsoadministering rifamycin or a rifamycin derivative, such as rifabutin ora rifabutin derivative. Such methods may, in particular, be employedwith drugs that have other harmful effects. For example, use of certainalkylating agents, such as topoisomerase inhibitors, increases the laterchances of leukemia in the patient. The chance of this adverse effectmay be lessened if lower doses of the alkylating agent may beadministered with the same therapeutic effect. Similarly, methods of thepresent disclosure may be used to reduce the amount of mitoticinhibitors administered, reducing the chance or amount of resultingperipheral nerve damage, or the methods may be used to reduce the amountof anti-tumor antibiotics administered, reducing the chance or amount ofresulting hearing damage. In the case of anti-tumor antibiotics forwhich there is a total lifetime dosage limit, methods of the presentdisclosure may allow a patient to be treated with the drug for a longertime, increasing life expectancy or improving quality of life. Methodsof the present disclosure may also allow amounts of somechemotherapeutics administered to remain sufficiently low as to allowthe patient to have children after cancer treatment. Methods of thepresent disclosure may further allow amounts of the chemotherapeuticsadministered to be lowered into a range where a drug approved for use inadults might also be used in children.

In an alternative embodiment in which the rifamycin or rifamycinderivative, such as rifabutin or rifabutin derivative directly inhibitsa cancer cell alone or in addition to causing drug-sensitization, thedosage and administration may be adequate to allow this inhibition. Inan example embodiment, it may consist of regular administration of anamount of the rifamycin or rifamycin derivative, such as rifabutin orrifabutin derivative, to maintain a certain level in the patient, theblood, a tissue, or a tumor. However, dosage amounts and theadministration schedule may be adjusted based on other components of thecomposition and their effects on drug availability in a patient, theintended mode of administration, the intended schedule foradministration, when other drugs are administered, any drug toxicityconcerns, and the patient's response to the drug.

Without limiting the compositions and methods of administrationdescribed herein, in one embodiment, rifamycin or a rifamycinderivative, sush as rifabutin or a rifabutin derivative, may exhibit itsdrug-sensitization effect on a cancer cell by directly or indirectlyinhibiting an efflux pump, such as the ATP-binding cassette sub-family Bmember 1 (ABCB1) pump. This glycoprotein is found in the cell membraneand actively transports certain chemotherapeutics, such as doxorubicin,out of cancer cells, reducing efficacy of the drug. By inhibiting thispump, the amount of chemotherapeutic present in a cancer cell can beincreased and thus the killing effect on the cancer cell may beincreased.

According to one embodiment of the present disclosure, rifabutin andrifabutin derivatives suppress ABCB1 activity, increasing the effectiveamount of a chemotherapeutic within a cancer cell.

Also, without limiting the compositions and methods of administrationdescribed herein, in one embodiment, rifamycin or a rifamycinderivative, such as rifabutin or a rifabutin derivative, may exhibit itsdrug-sensitization effect on a cancer cell by acting on the Akt (proteinkinase B)/14-3-3ζ/mitochondrial electron transport chain (ETC)/reactiveoxygen species (ROS) signaling network within a cell. An example of howthe rifamycin or rifamycin derivative can effect this pathway in adrug-resistant cancer cell is shown in FIG. 1. In this example, aCHOP-resistant cancer cell, such as a CHOP-resistant diffuse largeB-cell lymphoma (DLBCL) cell, undergoes cellular changes such that Aktis constitutively activated. This constitutively activated Aktphosphorylates mitochondrial GSK-3β. This phosphorylated GSK-3β thenbinds the 14-3-3ζ protein, rendering the GSK-3β unavailable to bind tomitochondrial ETC Complex 1. GSK-3β binding to ETC Complex 1 inhibitsthe complex activity, so the overall result of constitutive Aktactivation is that ETC Complex 1 is not inhibited when it otherwiseshould be. Downregulation of Complex 1 activity by GSK-3β can lead toincreased electron leakage from the ETC, resulting in increases in ROS.

ETC Complex 1 acts to reduce the amount of electron spillage from theETC during mitochondrial activity. Electrons spilled in such a mannerreact with oxygen to produce reactive oxygen species (ROS). Thus,increased ETC Complex 1 activity and the resultant reduction in electronleakage decrease the amount of ROS in the cell. Low levels of ROS maylead to an intracellular environment that inhibits the ability ofchemotherapeutics such as CHOP to induce cancer cell death by apoptosis.Thus, one effect of constitutive Akt activation is a decrease in ROS,making the cancer cell harder to kill.

According to one embodiment of the present disclosure, rifamycin or arifamycin derivative, such as rifabutin and rifabutin derivativesuppress ETC Complex 1 activity, restoring it to a more normal level. Asa result, more ROS are present in the cell and the cellular environmentis restored to one in which CHOP may once again induce cell death viaapoptosis.

A similar effect may be seen with other chemotherapeutics or other drugswhose efficacy relies on a cellular environment with minimum amount ofROS or other factors (such as other intracellular chemicals, proteins,or conditions) resulting from a minimum amount of ROS in the cell.

As a result of this effect on the Akt/14-3-3ζ/ETC/ROS network, thepresent disclosure also includes methods of inducing drug-sensitizationin a cancer cell by administering an amount of rifabutin or rifabutinderivative sufficient to decrease activity of ETC Complex 1 or increasecellular levels of ROS. In particular, the disclosure includes methodsof administering an amount of rifabutin or rifabutin derivativesufficient to increase cellular levels of ROS to an amount sufficient toallow a drug to which a cancer cell is sensitized to kill, reduce thegrowth of, or negatively affect the cancer cell.

Although the above example relates to cancer cells that have becomeresistant to a drug due to abnormal Akt activity, the same methods areapplicable to cancer cells that exhibit low ROS levels for otherreasons. Furthermore, the same methods may be used fordrug-sensitization in cancer cells that have no ROS abnormality byincreasing ROS to an abnormal level if the cancer cells then becomesensitive to the drug at the abnormal ROS level.

Effects mediated by ROS described above may, in particular, be mediatedby superoxide species and superoxide species may be the particular formof ROS affected.

Although some drug-sensitization or cancer cell inhibition effects maybe mediated by the ROS pathway, compositions and methods of the presentdisclosure may act via other cellular pathways alternatively to or inaddition to the Akt/14-3-3ζ/ETC/ROS network. This may be particularlytrue with respect to drug-sensitization to chemotherapeutics thatoperate in a different manner than CHOP. For example, ROS may affect themitochondrial-directed Bcl-2 apoptosis pathway as well. Furthermore, theeffect of rifabutin on ROS induction has been shown to be very rapid,whereas the effect on Akt has been shown to take at least 18 hours.Accordingly, it appears likely that an initial ROS induction event mayoccur, followed by a secondary downstream effect downregulating Akt. InCHOP-resistant cells, Akt is constitutively active thereby increasingComplex I activity resulting in decreases in ROS. Induction of ROS bycompositions and methods of the present disclosure will further promotedrug sensitivity in the resistant cancer cell by downregulating the Aktpathway.

Again without limiting the compositions and methods of administrationdescribed herein, in one embodiment, rifamycin or a rifamycinderivative, such as rifabutin or a rifabutin derivative, may exhibit itsdrug-sensitization effect on a cancer cell by mobilizing calcium withinthe cell. Increased calcium mobilization correlates with increased ROSamounts. Drug-sensitive cells often exhibit both increased levels ofcalcium and increased ROS levels as compared to drug-resistant cells.Typically, ROS levels rise first in such cells, followed by calciummobilization. Accordingly, rifamycin or a rifamycin derivative, such asrifabutin or a rifabutin derivative, may directly inhibit efflux pumpactivity, which then causes a burst of ROS followed by calciummobilization.

According to one embodiment of the present disclosure, rifamycin or arifamycin derivative, such as rifabutin or a rifabutin derivative, mayinhibit a cancer cell through more than one activity. For instance, itmay both decrease efflux pump activity and increase ROS. In certainembodiments, these multiple activities may have synergistic effects.

Further without limiting the compositions and methods of administrationdescribed herein, the compositions and methods may prevent or reducemetastasis. Metastasis from solid tumors is a complex, multistep processwhereby cancer cells must breach the basement membrane and migrate awayfrom the primary tumor environment to invade the surrounding stroma andenter the vasculature directly or via the lymphatics. The cancer cellsmust then also invade another area of the body. Rifamycin or a rifamycinderivative, such as rifabutin or a rifabutin derivative, may prevent orreduce metastasis by preventing or reducing any of these movements oractivities of the cancer cells. Rifamycin or a rifamycin derivative,such as rifabutin or a rifabutin derivative, may also decrease thelevels of metastasis-associated cellular factors in or around cancercells. Such factors include matrix metalloproteinase (MMP) 2 or otherMMP family members and vascular endothelial growth factor (VEGF). MMPfamily members are involved in the breakdown of extracellular matrix indisease processes such as metastasis. VEGF is an important signalingprotein involved in both vasculogenesis (the formation of thecirculatory system) and angiogenesis (the growth of blood vessels frompre-existing vasculature).

Determining Appropriate Cancer Targets

The present disclosure also provides a method of determining whether acancer cell is likely to be resistant to chemotherapeutics or experiencean increase in ROS or drug-sensitization in response to rifamycin or arifamycin derivative, such as rifabutin or a rifabutin derivative, or iftreatment with such a composition is having an effect on a cancer cell.In such a method. ROS, such as superoxide species, may be measured incancer cells of the same type. If ROS is abnormally low compared to ROSlevels previously measured in cancer cells of the same type (e.g. 3-10fold lower), then the cancer cell may be more likely to not respond to achemotherapeutic than cancer cells with higher ROS levels. The cancercell may also exhibit an increase in ROS or be sensitized to a drug inresponse to rifamycin or a rifamycin derivative, such as rifabutin or arifabutin derivative, so such a composition may be administered to thecancer cell along with a drug to which a cancer cell is sensitized toinhibit the cancer cell. If the patient has been treated with rifamycinor a rifamycin derivative, such as rifabutin or a rifabutin derivative,and ROS levels are normal for that type of cancer cell, higher than inprevious measurements from that patient, or higher than normal for thetype of cell from which the cancer is derived, then the treatment islikely successful and should be continued.

Alternatively, rather than measure ROS directly, an indicator of ROSlevels may be measured. In a specific embodiment, ROS may be measuredusing ROS stains.

In another embodiment, the amount of ROS (or indicator of ROS levels) incancer cells of a certain type may be measured and, if below a certainthreshold, rifamycin or a rifamycin derivative, such as rifabutin or arifabutin derivative, may be administered to a cancer cell of the sametype along with a drug to which a cancer cell is sensitized to inhibitthe cancer cell.

In specific embodiments, ROS-related measurements may be made by anyconventional methods. For example, ROS-related measurements may be madeon a biopsy, resection or aspirant of a tumor or cancer-bearing tissue,a blood sample, or cancer cells isolated by other means. Measurementsare compatible with presently known methods of obtaining cancer cellsfrom patients and are expected to be similarly compatible withadditional methods developed in the future.

EXAMPLES

The following examples are provided to further illustrate specificembodiments of the disclosure. They are not intended to disclose ordescribe each and every aspect of the disclosure in complete detail andshould be not be so interpreted. Unless otherwise specified,designations of cells lines and compositions are used consistentlythroughout these examples.

Example 1 Drug-Sensitization of CHOP-Resistant NHL Cell Lines

Several human cell lines were utilized as in vitro models of NHL,including the CRL2631 line obtained from the American Type CultureCollection (ATCC). CRL2631 was established from peripheral bloodleukocytes (PBL) of a patient with DLBCL. CHOP-resistant NHL cell lines(designated G3) were generated by repeated cycles of on-off treatmentswith CHOP, a treatment protocol that is similar to clinical regimens.

The effects of rifabutin on cell growth of both CHOP-sensitive (CRL2631)and CHOP-resistant (G3) cells in the presence or absence of CHOP areshown in FIG. 2A. A reduction in cell growth is demonstrated by areduction in fluorescence emitted by the cell growth indicator dye,resazurin.

Rifabutin was confirmed to have drug-sensitization activity inclinically derived CHOP resistant cell lines. As shown in FIG. 2A, CHOPinhibited growth of CHOP-sensitive (CRL2631) cells but had little effecton G3 cells. Rifabutin did not affect the growth of cells in the absenceof CHOP, indicating low toxicities (FIG. 2A). Rifabutin enhanced thesensitivity of CHOP-sensitive cells to CHOP as shown in FIG. 2B,relative to a control drug. FIG. 2C shows similar effects in anotherCHOP-resistant NHL line.

Example 2 Toxicity

Doxorubicin and rifabutin or its derivatives RTI-79 and RTI-176 wereapplied to primary human fibroblasts to determine comparativecytotoxicity. Results are presented in FIG. 3A and FIG. 3B anddemonstrate that rifabutin and the analogs are not toxic to normalcells.

To further test safety of rifabutin and its derivatives, rifabutin andrifabutin derivates RTI-79 and RTI-81 were administered as an adjuncttherapy to doxorubicin (DOX).

Swiss mice were dosed with levels equal to and exceeding that ofintended doses. Swiss mice were given repeated weekly oral doses ofrifabutin at 180 mg/kg, RTI-79 at 250 mg/kg or RTI-81 at 30 mg/kg inconjunction with intravenous 3.3 mg/kg DOX. No overt toxicity or weightloss was seen over several weeks time. Further, no significantdifferences between mice treated with RTI-79 with or without DOX wereobserved after both histological analysis of heart tissue by hematoxylinand eocin (H&E) and analysis of blood and serum for complete blood countand manual differential. Intravenous rifabutin or RTI-81 were also givenrepeatedly both at 75 mg/kg in conjunction with intravenouslyadministered 3.3 mg/kg DOX and no overt toxicity or weight loss was seenover several weeks time. Further data in Example 4 below shows treatmentefficacy using less than one-fifth the above oral dose of 33 mg/kgrifabutin with intravenous 3.3 mg/kg doxorubicin.

Example 3 Drug-Sensitization of CHOP-Resistant Lymphoma Cells from DogModel

A single lymphoma aspirate from a dog with CHOP-resistant lymphoma wastested for responsiveness to CHOP in the presence or absence ofrifabutin. CHOP-responsiveness was measured by a decrease influorescence signal generated by resazurin. FIG. 4 shows that growth ofaspirated lymphoma cells was resistant to CHOP at doses up to 640 ng/ml,but significant growth inhibition was observed at a dose of 1280 ng/mlCHOP. The inclusion of 5 μM rifabutin significantly enhanced thesensitivity of the aspirated lymphoma cells to CHOP such thatsignificant growth inhibition was observed at 320 and 640 ng/ml CHOP.Rifabutin had no effect on cell viability in the absence of CHOP.

Example 4 Drug-Sensitization In Vivo

In a first efficacy study, 6-8 week old female SCID mice (7 mice pertreatment arm) were injected subcutaneously on both flanks with 1×10⁷ G3CHOP-resistant NHL cells. Once palpable tumors (about 50-100 cc size)appeared, therapies (CHOP or CHOP+rifabutin) were started. CHOP wasadministered at the maximum tolerated dose (cyclophosphamide, 40 mg/kgi.v.; doxorubicin, 3.3 mg/kg i.v.; vincristine, 0.5 mg/kg i.v.; andprednisone, 0.2 mg/kg orally daily for 5 d) weekly for 3 weeks.Rifabutin in an amount of 100 mg/kg was administered on the day of eachCHOP treatment and 24-hours later by gavage. Mouse body weight and tumorsize were monitored every two days and tumor size measured by caliper.The tumor volume formula (L*W*W)/2 was used to calculate tumor mass.

The overall tumor burden per mouse was much lower in mice that receivedCHOP+rifabutin than for those receiving CHOP only treatments. CHOPtreatment alone of the SCID mice harboring subcutaneous G3 lymphomasresulted in relatively fast tumor growth, as compared to tumors inCHOP+rifabutin treated mice (FIG. 5). The dosage of rifabutinadministered had little or not toxicity in the mice. Control miceinjected with CRL2631 cells, in contrast, exhibited a marked decrease intumor growth in response to CHOP alone (FIG. 6).

A second efficacy study was conducted where mice were treated before theappearance of palpable tumors. In that experiment, one week aftertransplantation of CHOP-resistant G3 cells, one group (7 mice) wastreated with CHOP-only and a second group (8 mice) was treated withCHOP+rifabutin. One week later, mice received a second treatment andtumors began to appear in the CHOP-only group. The two treatment groupsdiffered not only in the tumor size but also in the number of tumorsdeveloped. More tumors appeared and grew at a significantly higher ratein CHOP-only mice compared to CHOP+rifabutin mice. The CHOP onlytreatment group developed tumors at 12 of 14 (85.7%) injection sites.The CHOP+rifabutin treatment group developed fewer tumors at only 6 of16 (37.5%) injection sites. In a separate experiment, SCID micedeveloped G3 tumors at 35 of 42 (83.3%) injection sites when receivingno treatment; this is similar to the CHOP only treatment group.Significance was analyzed by the T test yielding a highly significantdifference between the means of the tumor burdens of the two groups(p<0.01) at Day 7. Thus, rifabutin actually reduces the tumor take ratewhich could translate into more complete responses when humans aretreated early with this combination.

A third study was conducted in which mice injected with CHOP-resistantG3 cells received reduced dosages of CHOP in combination with 33 mg/kgrifabutin. CHOP+rifabutin was administered weekly beginning one weekpost-inoculation. Control mice were given no CHOP or rifabutin. Tumorload was significantly less in mice that received even reduced CHOPdosages as compared to untreated mice, demonstrating that rifabutin mayallow the use of lower dosages of CHOP without a significant decrease intherapeutic effect (FIG. 7).

A fourth efficacy study was conducted using DOX in combination with therifabutin derivative RTI-81. SCID mice were injected with CHOP-resistantG3 cells in the same manner as the first efficacy study above.Treatments began 2 weeks post-inoculation and were administered twiceweekly. DOX was given at 3.3 mg/kg iv and RTI-81 was given at 10 mg/kgby gavage. A statistically significant difference in average life-spanis seen when mice were treated with doxorubicin and RTI-81 as comparedto DOX alone. Mice receiving doxorubicin+RTI-81 lived 27% longer thanthose receiving doxorubicin only (X²=8.6 p=0.00336 (dof=1)) (FIG. 8).Respective mean and median lifespans for each group were: 42.6, 42 and34.6, 33. Mice treated with doxorubicin only were 10.37 times as likelyto die before those treated with doxorubicin+RTI-81. Cox proportionalhazard ratio was 0.0964 with a likelihood ratio of 7.24 (p=0.00714(dof=1, n=15).

In a fifth efficacy study, we generated xenografts of the human ovariancancer cell line SK-OV-3, a cell line considered doxorubicin-resistant,by bilateral subcutaneous (s.c.) injection of 1×10⁷ tumor cells toestablish localized tumors in 6-8 week old female SCID mice. Usingrifabutin co-administered with DOX, in vivo efficacy was assessed. Oncetumor volumes were at least 75 mm³ and showed consistent growth rates,therapies (DOX only 3.3 mg/kg i.v. or DOX 3.3 mg/kg i.v.+rifabutin 25mg/kg oral) were started. Cycles of Dox or Dox+rifabutin were given oncea week for 4 cycles. This cyclical dosing scheme of mouse models hasprecedent in the literature and is intended to mimic the cycles ofDOXIL® (Centocor Ortho Biotech Products. LP, Raritan, N.J.) given in theclinic. Rifabutin was administered on the day of each DOX treatment andby gavage. Mouse body weight and tumor size were monitored. As shown inFIG. 9, after 19 days treatment, average tumor volumes were 587 mm³ forthe DOX-only treatment group, and 348 mm³ for the DOX+rifabutin group.This is a 40% reduction in tumor size for the DOX+rifabutin group.

In a sixth efficacy study, we generated xenografts of multi-drugresistant ovarian cancer cell line (NCI/ADR-RES) by implantation ofNCI/ADR-RES cell xenografts in the left and right flanks of nude mice,resulting in two tumors per mice. In vivo efficacy of RTI-79 wasassessed by co-administration with DOXIL®. Once tumor volumes were atleast 90 mm³ and showed consistent growth rate, therapies (DOXIL®) only7 mg/kg i.v. or DOXIL® 7 mg/kg i.v.+RTI-79 25 mg/kg oral) were started.Cycles of DOXIL® or DOXIL®+RTI-79 were given every week for six cycles.RTI-79 was administered by oral gavage 24 and 48 hours after each DOXIL®administration. Tumor size was monitored. As shown in FIG. 10, after 41days the tumor volume in the RTI-79-treated mice was 66% lower than inmice receiving only DOXIL®.

In a seventh efficacy study, we generated xenografts of multi-drugresistant ovarian cancer cell line (NCI/ADR-RES) by implantation ofNCI/ADR-RES cells xenografts in the left and right flanks of nude mice,resulting in two tumors per mouse. In vivo efficacy of RTI-79 wasassessed by co-administration with DOXIL®. Therapies were DOXIL® only 7mg/kg i.v. or DOXIL® 7 mg/kg i.v.+RTI-79 25 mg/kg oral. Cycles of DOXIL®or DOXIL®+RTI-79 were given every week for six cycles. RTI-79 wasadministered by oral gavage 24 and 48 hours after each DOXIL®administration. Tumor size was monitored. As shown in FIG. 11, after 46days the tumor volume in the RTI-79-treated mice was 55% lower than inmice receiving only DOXIL®. Furthermore, tumor volume in RTI-79-treatedmice was reduced by 50% during the course of the study.

Example 5 Sensitization to CHOP Using Other Rifabutin Derivatives

Several compositions of the present disclosure were tested and theireffects on cell growth were measured. A reduction in cell growth isdemonstrated by a reduction in fluorescence emitted by the cell growthindicator dye, resazurin. Compositions were tested on CHOP-resistant G3NHL cells that had been cultivated in RPMI medium for five days. Priorto assay, the cells were counted by haemocytometer and cellconcentration standardized to 625,000 cells/ml. Test drugs weresolubilized in 100% DMSO and then diluted to final assay concentrationwith 0.1M phosphate buffered saline (PBS) and a final DMSO concentrationof 0.5%. Cells were added to assay plates containing the test drugs(rifabutin+148 ng/ml, 74 ng/ml, 37 ng/ml, or 0 ng/ml doxorubicin) andallowed to incubate for 96 hours at 37° C., and 5% CO₂. The metabolicdye rezasurin was added to the wells of the assay plate at a finalconcentration of 20 μg/ml and the plates were incubated for anadditional 24 hours. The plates were then read in a BMG Polarstar platereader at wavelength (573-605) and the data plotted as OD versusincreasing dilutions (i.e., decreasing total amounts) of rifabutinderivative concentration.

Results of tests were performed on G3 cells to compare the effects ofrifabutin and certain rifabutin derivatives on cell growth in thepresence or absence of 1 μM doxorubicin (DOX) are presented in Table 2,which indicates the IC₅₀s for selected rifamycin analogs on lymphomacell line G3.

TABLE 2 IC₅₀s for selected rifamycin analogs on lymphoma cell line G3.IC₅₀ (μM) with Fold increase in potency Analog IC₅₀ (μM) doxorubicin (1μM) over DOX alone Doxorubicin 2.36 NA NA Rifabutin >64 .25 9.4RTI-51 >64 3.3 0.7 RTI-53 >64 11.8 0.2 RTI-78 58 0.08 29.5 RTI-79 431.14 2.1 RTI-81 >64 0.3 7.9 RTI-82 >64 3.5 0.7 RTI-102 >64 0.95 1.5RTI-174 51 0.64 3.7 RTI-175 >64 1.06 2.2 RTI-176 >64 0.45 5.2 RTI-181 520.43 5.5 RTI-182 62 1.2 2.0 RTI-183 >64 5.19 0.5

Example data for RTI-79 and rifabutin is shown in FIG. 12. Example datafor RTI-176 and rifabutin is shown in FIG. 13. Example data for RTI-81and rifabutin is shown in FIG. 14. Example data for interaction ofrifabutin and doxorubicin on CRL2631 cells is shown in FIG. 15. Exampledata for interaction of RTI-79 and doxorubicin on CRL2631 cells is shownin FIG. 16. These results establish that a variety of rifabutinderivatives are similarly effective at restoring doxorubicin sensitivityto CHOP-resistant cells.

Example 6 Drug-Sensitization of Multiple Cell Lines

The ability of rifabutin and rifabutin derivatives to causedrug-sensitization to doxorubicin in multiple types of cancer cells wasinvestigated by performing experiments similar to those described above.In these experiments, the following cell lines were used: CHOP-resistantNHL cell line G3, CHOP-sensitive NHL cell line CRL2631, the multi-drugresistant sarcoma cell line MES-SA-Dx5; multi-drug-resistant breastcancer cell line MDA-MB-231, multi-drug resistant ovarian carcinoma cellline SK-OV3, multi-drug resistant ovarian cancer cell line NCI/ADR-RES,drug-sensitive ovarian cancer cell line OVCAR-5, and multi-drugresistant ovarian cancer cell line OVCAR-3. Results are presented inTable 3.

TABLE 3 Magnitude of potentiation observed with rifamycin analogs incombination with doxorubicin Cancer RTI- RTI- RTI- RTI- Type Tissue CellLine RBT 51 53 79 81 Lymphoma B cells G3 +++ ++ +++ Lymphoma B cells CRLCRL2631 Sarcoma Uterus MES-SA- ++ ++ ++ Dx5 Carcinoma Breast MDA- +++ +MB-231 Carcinoma Ovarian SK-OV3 + + + +++ +++ Carcinoma OvarianOVCAR-3 + + + Carcinoma Ovarian OVCAR-5 + Carcinoma Ovarian NCI/ADR-++ + RES Cancer RTI- RTI- RTI- RTI- Type Tissue Cell Line RBT 82 102 174175 Lymphoma B cells G3 +++ ++ ++ ++ Lymphoma B cells CRL CRL2631Sarcoma Uterus MES-SA- ++ + Dx5 Carcinoma Breast MDA- +++ +++ MB-231Carcinoma Ovarian SK-OV3 + +++ + +++ Carcinoma Ovarian OVCAR-3 + +Carcinoma Ovarian OVCAR-5 ++ Carcinoma Ovarian NCI/ADR- + ++ RES CancerRTI- RTI- RTI- RTI- Type Tissue Cell Line RBT 176 181 182 183 Lymphoma Bcells G3 +++ +++ +++ + Lymphoma B cells CRL CRL2631 Sarcoma UterusMES-SA- ++ Dx5 Carcinoma Breast MDA-MB- +++ ++ 231 Carcinoma OvarianSK-OV3 + +++ +++ ++ Carcinoma Ovarian OVCAR-3 + + + Carcinoma OvarianOVCAR-5 + Carcinoma Ovarian NCI/ADR- RES RBT = rifabutin; + potentiationbetween 1.2 to 2.0 fold increase; ++ potentiation between 2.1 to 5 foldincrease; +++ potentiation greater than 5 fold increase

Example data for rifabutin or RTI-82 on MDA-MB-231 cells is presented inFIG. 17. Example data for rifabutin or RTI-79 with or withoutdoxorubicin on SK-OV3 cells is presented in FIG. 18. Example data forrifabutin or RTI-81 on MES-SA-Dx5 cells is presented in FIG. 19. Exampledata for interaction of rifabutin or RTI-79 and doxorubicin on ADR-REScells is shown in FIG. 20. Example data for interaction of RTI-79 anddoxorubicin on MOLT-4 cells is shown in FIG. 21. Example data for theinteration of rifabutin or RTI-79 and doxorubicin on ovarian carcinomaOVCAR-8 cells is shown in FIG. 22. These results establish thatrifabutin and rifabutin analogs are able to induce drug-sensitizationfor a variety of types of cancer.

Example 7 Sensitization to Various Chemotherapeutics Using Rifabutin andRifabutin Derivatives

Similar tests were performed to compare the effects of rifabutin andcertain rifabutin derivatives on cell growth in the presence or absenceof various chemotherapeutics on various cell lines. Chemotherapeuticsinclude: the targeted therapy bortezomib (Velcade®), the pyrimidineantagonist gemcitabine, the platinum drug cis-platin, the anti-tumorantibiotic actinomycin D, the anti-tumor antibiotic apicidin, thetopoisomerase I inhibitor camptothecin, the anti-tumor antibioticdoxorubicin, the mitotic inhibitor vinblastine, the nitrogen mustardalkylating agent melphalan, the hormonal agent tamoxifen, the folateantimetabolite methotrexate, the toposimerase II inhibitor etoposide,phenoxodiol, the antibiotic rapamycin, and menadione. Additional celllines used include: ovarian cancer OVCAR-8. T lymphoblastoid leukemiaMOLT-4, dexamethasone-resistant multiple myeloma MM.1R, myeloid leukemiacells HL-60, osteosarcoma cells U-2 OS, and myeloma RPMI 8226. Resultsare shown in Table 4.

TABLE 4 IC₅₀s for selected cancer cell lines and clinically relevantcancer therapeutics in interaction with Rifabutin IC₅₀ Fold (μM)increase IC₅₀ with in Cancer type Cell line Therapeutic drug (μM)Rifabutin potency Diffuse large B G3 Doxorubicin 2.36 0.25 9.4 celllymphoma Diffuse large B G3 Vinblastine 8.00 1.00 8.0 cell lymphomaDiffuse large B G3 Mitoxantrone 0.46 0.04 11.5 cell lymphoma Diffuselarge B CRL2631 Doxorubicin 0.35 0.12 2.9 cell lymphoma Ovariancarcinoma OVCAR-3 Menadione >32 10.88 >7.9 Ovarian carcinoma OVCAR-5Velcade 017 0.08 2.1 Ovarian carcinoma OVCAR-8 Mitoxantrone 14.0 3.0 4.7Ovarian carcinoma SK-OV3 Mitoxantrone >32 12.28 >2.6 Ovarian carcinomaADR-RES Doxorubicin >32 6.59 >4.9 Leukemia MOLT-4 Doxorubicin 0.03 0.013 Leukemia MOLT-4 Actinomycin D 0.04 <0.008 >5 Breast Cancer MDA-MB-231Gemcitabine >32 4.83 >6.6 Multiple myeloma MM.1R Camptothecin 1.13 0.33.8 Multiple myeloma MM.1R Menadione 4 2 2 Myeloid leukemia HL-60Paclitaxel 0.4 0.2 2 Uterine Sarcoma MES-SA-Dx5 Actinomycin D 0.03 0.013 Osteosarcoma U-2OS Mitoxantrone 0.14 0.06 2.3 Myeloma RPMI 8226Paclitaxel 1.0 0.89 1.1

Example data for interaction of rifabutin with actinomycin D onMES-SA-Dx5 cells is shown in FIG. 23. Example data for interaction ofrifabutin with menadione on MM.1R cells is shown in FIG. 24. Exampledata for interaction of rifabutin and mitoxantrone on U-2 OS cells isshown in FIG. 25. Example data for interaction of rifabutin andgemcitabine on MDA-MB-231 cells is shown in FIG. 26. Example data forinteraction of rifabutin with paclitaxel on HL-60 cells is shown in FIG.27. Example data for interaction of rifabutin and camptothecin onOVCAR-8 cells is shown in FIG. 28. These results demonstrate the abilityof rifabutin and rifabutin derivatives to induce drug-sensitivity for awide variety of chemotherapeutics in a wide variety of cancers.

Example 8 Prevention of the Emergence of CHOP Resistance

The ability of rifabutin to prevent the emergence of CHOP-resistance wasdetermined by treating CHOP-sensitive CRL2631 cells with either CHOPalone or CHOP+rifabutin for one week. Following treatment, the cellswere grown in the absence of CHOP, then their sensitivity to CHOP wasassayed by retreatment with CHOP, followed by counting of viable cells.Results are shown in FIG. 29. Rifabutin was able to significantlyrepress the emergence of CHOP-resistant cells at both half (0.5×) andfull (1×) doses of CHOP. A 1× CHOP dose in this experiment correspondsto final concentrations of the following components: 0.83 μM4-hydroxycyclophosphamide [4HC, a pre-activated form ofcyclophosphamide], 0.057 μM doxorubicin, 0.01 μM vincristine, and 0.186μM prednisone.

Example 9 Effects of Rifabutin and Rifabutin Derivatives on ROS

A Western blot of CHOP-sensitive (CRL2631) or CHOP-resistant (G3)lymphoma cells revealed that Akt, phosphorylated Akt, and 14-3-3ζ levelswere consistent with the model proposed in FIG. 1 (FIG. 30A) in that Aktwas markedly more active in CHOP-resistant G3 cells than in CRL2631. Themodel was further confirmed by treatment of CHOP-resistant (G3) cellswith Akt Inhibitor VIII, which caused a dose-dependent reversal of CHOPresistance (FIG. 30B). The inhibitory effect of Akt inhibitor VIII onthe expression of phosphorylated Akt and total 14-3-3ζ protein wasconfirmed by Western blot (FIG. 30C).

Additional studies further confirmed the model of FIG. 1 bydemonstrating that CHOP-sensitive (CRL2631) cells make more ROS than doCHOP-resistant (G3 cells) (FIG. 31). Furthermore, CHOP increased ROS inCHOP-sensitive (CRL2631) cells, but not in CHOP-resistant (G3) cells(FIG. 31).

Examination of CHOP-sensitive CRL2631 cells revealed that these cellsinclude two distinct populations, a low-ROS population and high-ROSpopulation (FIG. 32). When these populations were separated, the low-ROSpopulation proved more resistant to CHOP than high-ROS population (FIG.33). However, this low-ROS cell population was sensitized to CHOP byrifabutin (FIG. 34). Rifabutin also rapidly induces ROS inCHOP-resistant (G3) cells (FIG. 35).

Overall, these results demonstrate that, at least in the CRL2631lymphoma cell line and cell lines derived therefrom, CHOP-resistance ismediated by ROS levels and that rifabutin and rifabutin derivativesdecrease CHOP-resistance by increasing ROS.

Example 10 Rifabutin and RTI-79 Decrease Drug Efflux and MobilizeCalcium

Rifabutin and its derivatives, such as RTI-79, showed clear inhibitionof efflux pumps in NCI/ADR-RES and G3 cells when tested in calcein-AMassays. This inhibitory effect was unambiguously due to inhibition ofABCB1 pumps. The difference in pump activity between ADR-RES cells andits drug-sensitive parental strain, OVCAR-8, may be seen in FIGS. 34Aand 34B

It is known that mitigation of ABCB1 activity will lead to moreeffective accumulation of doxorubicin in cells (Shen, F., Chu, S.,Bence, A. K., Bailey, B., Xue, X., Erickson, P. A., Montrose, M. H.,Beck, W. T., and Erickson. L. C. (2008). Quantitation of doxorubicinuptake, efflux, and modulation of multidrug resistance (MDR) in MDRhuman cancer cells. J Pharmacol Exp Ther 324, 95-102). Thus theinhibition on ABCB1 by RTI-79 directly contributes to its potentiatingdoxorubicin toxicity on these drug-resistant cells. This was confirmedby testing with additional rifabutin derivatives. As shown in FIG. 36,the stronger inhibitors of ABCB1, also better re-sensitizeddrug-resistant cells. RTI-79 was the strongest inhibitor as well as bestre-sensitizer.

Doxorubicin-sensitive (OVCAR8 ovarian) and Doxorubicin-resistant (G3lymphoma; ADR-RES ovarian) cells were treated for 2 hrs with 10 uMRTI-79, p-glycoprotein (P-gp) inhibitors (reserpine, elacridar), orcontrol drugs (DMSO, carboxin, nifazoxidine). Cells were then stainedwith the fluorescent ROS indicator, CellROX, and subjected to flowcytometry to quantitate total intracellular ROS. As FIG. 38 shows,RTI-79 induced ROS in ovarian carcinoma and lymphoma cell lines, as didthe MDR/P-gp inhibitors, reserpine, elacridar. This suggests that RTI'sability to induce ROS was the result of inhibition of efflux pumps. Thedegree of ROS induced by RTI-79 and P-gp inhibitors was much greater inthe doxorubicin-resistant ADR-RES and G3 cell lines than in thedoxorubicin-sensitive OVCAR8 cell line. Control drugs established thatthis effect is specific to MDR/P-gp inhibitors and RTI-79.

The intracellular origin of RTI-induced ROS was determined by stainingADR-RES cells with the red fluorescent ROS indicator, CellROX(Invitrogen), and visualizing where the ROS was concentrated by confocalmicroscopy. Results are presented in FIG. 39. Mitochondria werelocalized by infecting cells for 24 hrs with the BacMAM mitotrackerbaculovirus, which expresses a GFP fused to a mitochondria localizationsignal. Nuclei were stained with the blue DAPI stain. There was a goodco-localization of red CellROX staining with the green GFP mitotracker,indicating that the ROS were originating from the mitochondria.

The electron transport chain (ETC) is known to be a primary generator ofROS in the cell. Most of the ROS is generated by Complexes I and III ofthe ETC. Inhibition of Complex I results in electrons piling up andleaking to react with oxygen to produce ROS. The effects of a Complex Iinhibitor (rotenone) and a Complex III inhibitor (antimycin A) on ROSlevels in the cell were tested and results are shown in FIG. 40.Specifically, Dox-resistant G3 lymphoma cells were treated 10 uM RTI-79,BAPTA-AM (cell permeable calcium chelator), verapamil (a calcium channelblocker and P-gp inhibitor), a Complex I inhibitor (Rotenone), a ComplexIII inhibitor (antimycin A), or control drugs (oxaloacetate, carboxin,nifazoxinide). Rotenone, but not antimycin A, induced ROS, suggestingthat RTI-79-induced and efflux pump inhibitor-induced ROS originate atComplex I of the ETC.

MDR/P-gp activity is closely associated with calcium status in the cell,so calcium modulators were tested for effects on ROS. As shown in FIG.40, both a cell-permeable calcium chelator (BAPTA-AM) and a calciumchannel blocker (and efflux pump inhibitor) induced ROS in G3 cells. Asshown in FIGS. 41A and 39B, P-gp inhibitors (Reserpine, Elacridar)induced ROS relative to control drugs (Carboxin, Nifazoxinide). As shownin FIG. 41B, a P-gp inhibitor (Elacridar) induced calcium in a similarmanner as RTI-79, indicating connections between calcium, ROS, andefflux pump activity in the mechanism of action of RTIs. Because calciummodulators induced ROS, testing was performed to investigate whetherRTI-induced ROS was associated with calcium mobilization indoxorubicin-sensitive and doxorubicin-resistant cells and in resistantcells treated with RTI-79. Relatively Dox-sensitive lymphoma (CRL2631,10S, WSU) and ovarian carcinoma (OVCAR8) and more Dox-resistant lymphoma(G3R, 10R, WSUR) and ovarian carcinoma (ADR) were treated with 10 uMDMSO for 2 hrs. Dox-resistant cells were also treated with 10 uM RTI-79for 2 hrs (G3R+RT179; 10R+RTI-79, WSUR+RTI-79). Cells were co-stainedwith the cell-permeable red fluorescent ROS indicator, CellROX, andcell-permeable green fluorescent calcium indicator. Fluo-4AM, and thensubjected to flow cytometry to quantitate changes in ROS and calciumlevels. As shown in FIG. 41C, levels of both ROS and calcium indoxorubicin-sensitive cells were much higher than in the resistantlines, and RTI-79 induced both ROS and calcium mobilization in resistantcells. Thus, the ability of RTI-79 to sensitize doxorubicin-resistantcells was closely correlated with the inhibition of efflux pumps,induction of ROS, and mobilization of calcium.

To determine whether increases in ROS led to calcium mobilization orcalcium mobilization resulted in ROS induction, a time course of RTI-79treatment of G3 cells monitoring ROS and calcium was conducted. Cellswere co-stained with the red fluorescent ROS indicator, CellROX, and thegreen fluorescent calcium indicator, Fluo-4AM for 30 minutes and treatedwith 10 uM RTI-79 for 0 to 30 minutes. All samples were analyzed at thesame time in flow cytometry. As shown in FIG. 42, increases in ROS wereseen as soon as 1 minute after exposure of cells to RTI-79 and graduallyincrease to 4 minute, level off to 5 minute, and then decrease after 6minute, followed by increases up to 15 minute. In contrast, calciummobilization did not occur until after 15 minutes of RTI-79 treatment,thus indicating that ROS levels increased first followed by calciummobilization.

RTI-79 might inhibit MDR/P-gp by inducing ROS, which then increasecalcium mobilization that then inhibits efflux pump activity.Alternatively. RTI-79 may first directly inhibit efflux pump activity,which then causes a burst of ROS followed by calcium mobilization. Todetermine which mechanism most likely involved, ADR-RES (Dox-resistant)and OVCAR8 (Dox-sensitive) ovarian carcinoma cells were transfected withsiRNA to knockdown efflux pumps to determine the effect on ROS andcalcium. Cells were then co-stained with CellROX and Fluo-4AM for 1hour. Some cells were treated with RTI-79 for 1 hour and controls (noRTI-79) were treated with DMSO. As shown in FIG. 43, knockdown of P-gpin ADR-RES cells led to increases in both ROS and calcium mobilization,and greatly enhanced the ability of RTI-79 to increase ROS and calciummobilization. As expected, the effect of downregulating efflux pumpactivity on ROS and calcium in OVCAR8 was much less than in ADR-RES, dueto the lower efflux pump activity in OVCAR8 cells. However, the degreeof induction of ROS and calcium mobilization by RTI-79 in P-gp knockdowncells (greater than 90% repression of P-gp expression) is much greaterthan what would be expected if the P-gp was the sole mechanism involvedin RTI-79-induced upregulation of ROS. Thus, is it likely that RTI-79acts not only to induce ROS and calcium mobilization through inhibitionof ROS, but also acts at a second target, namely Complex I, to induceROS.

Example 11 Preventing or Reducing Metastasis

The effects of rifabutin on cell invasion was assessed in a collageninvasion 3D assay. Increased interest in the use of 3D culture systemshas been motivated by accumulating evidence that 3D models betterreflect the microenvironment of tumors and metastases and moreaccurately predict therapeutic response in vivo compared withconventional 2D assays. A collagen invasion 3D assay allows the rapidand quantitative assessment of invasiveness and a means to screen fordrugs which alter the invasive phenotype of tumor cells. Malignant celllines with high metastatic potential in vivo show a higher rate ofinvasion than non-metastatic tumor cells and normal cells showed littleor no ability to penetrate the barrier.

The CHOP-resistant G3 cell line is much more invasive in a collageinvasion 3D assay than its CHOP-sensitive parent cell line (CRL2631).Collagen matrices (1 mg/ml) were prepared as previously described in Su.S. C., et al., Molecular profile of endothelial invasion ofthree-dimensional collagen matrices: insights into angiogenic sproutinduction in wound healing. Am. J. Physiol. Cell Physiol., 295(5):C1215-29 (2008), incorporated in material part by reference herein, withthe inclusion of either DMSO control or 10 uM Rifabutin (Rif). Cellswere allowed to invade for 24 hours. Culture medium was removed andcollagen gels containing invading cells were fixed in 3% glutaraldehydein PBS for 30 minutes. Gels were stained with 0.1% toluidine blue in 30%methanol for 10 minutes prior to destaining with water. Cell invasiondensity was quantified by counting fixed cultures under transmittedlight using an Olympus CK2 inverted microscope equipped with eyepiecesdisplaying a 10×10 ocular grid. For each condition, four random fieldswere selected and the number of invading cells per high power field(HPF) was counted manually at 10× magnification, corresponding to 1 mm²area.

Data are reported as mean number of invading cells per HPF (±S.D.) inFIG. 44. G3 cells were more invasive than CRL2631 cells. The inclusionof rifabutin in the collagen matrix reduced the amount of G3 invasion byup to 30%. Less of this effect was observed for CRL2631 cells.

A modified Boyden chamber assay was used as an independent method toevaluate rifabutin's ability to suppress invasion/metastasis. G3 andCRL2631 cells were grown in the presence of 10 μM rifabutin or dosevolume equivalent DMSO for 24 hours at 37° C. Cell invasion was assessedwith a Chemicon QCM Collagen Invasion Assay (Millipore). The assay is a96-well plate assay wherein each well is equipped with a suspendedinsert. Inserts contain an 8-micron membrane coated with a thin layer ofpolymerized collagen. Invading cells migrate through the collagen layerand attach to the bottom of the membrane. Cells were detached from themembrane and lysed prior to detection via CyQuant dye. Fluorescenceintensity is proportional to number of invading cells. As shown in FIG.45, the presence of rifabutin resulted in decreased relativefluorescence from 170,374 to 114,395 RLU in G3 cells. In CRL2631 cellsRLU decreases from 39,356 to 27,432 RLU in the presence of rifabutin(p<0.05).

The effect of RTI-79 treatment on the secretion of MMP2 and VEGF wasalso analyzed. Treatment with RTI-79 resulted in statisticallysignificant decreases in both MMP2 and VEGF in U2-OS osteosarcoma cellsin commercially available ELISA based assays. In U2-OS cells, MMP2 wasreduced from 22.4 ng/million cells to 10.5 ng/million cells (p<0.01)with the addition of 5 uM RTI-79. When evaluating the effects of RTI-79on VEGF, a decrease from 998 to 436 pg/million cells (p<0.01) wasobserved.

Example 12 Rifabutin Derivative Synthesis

The 3,4-cyclo-rifamycin (rifabutin) derivatives of the currentdisclosure made be prepared as shown in the schemes listed below.

Scheme 1 illustrates the general preparation of11-deoxo-11-imino-3,4-spiro-piperidyl-rifamycins (1c) and11-deoxo-11-amino-3,4-spiro-piperidyl-rifamycins (1d). The compounds of(1c) are synthesized by condensation of3-amino-4-deoxy-4-imino-rifamycin S (1a) with a substituted piperidoneor hexanon-type of ketone (1b) at a temperature range from 10° C., to70° C., in organic solvent, such as THF or ethanol, in the presence ofan excess of ammonium salt, such as ammonium acetate, in a sealedreaction tube. Reduction of 11-imino-rifamycin (1c) with reducingreagent, such as NaBH₄, in organic solvent, such as THF and EtOH at atemperature range from 0° C., to room temperature produces11-amino-rifamycin (1d). When the compound is RTI-35, the thioethercould be oxidized to sulfoxide (—SO—) or sulfone (—SO2-) depending uponthe ratio of compound 1c and oxidizing agents. When the compound isRTI-44, product is obtained by de-protection of Boc-protected-piperidineor Fmoc-protected-piperidine.

Scheme 2 illustrates the general preparation of3,4-spiro-piperidyl-rifamycins (2c) and11-deoxo-11-hydroxy-3,4-spiro-piperidyl-rifamycins (2d). The compoundsof (2c) are synthesized by condensation of3-amino-4-deoxy-4-imino-rifamycin S (1a) with a substituted piperidoneor hexanon-type of ketone (1b) at a temperature range from 10° C., to70° C., in organic solvent, such as THF or ethanol, in the presence orabsence of a catalyst, such as Zinc. Reduction of 11-oxo of rifamycin(2c) with reducing reagent, such as NaBH₄, in organic solvent, such asTHF and EtOH at a temperature range from 0° C., to room temperatureproduce 11-hydroxy-rifamycin (2d).

The intermediate of (1a) is commercially available or may be obtainedfrom the rifamycin S. The hexanon-type of ketone or 4-substitutedpiperidone (1b or 2b: Z═C, or O) is either commercially available or maybe prepared by known procedures. The 4-oxo-piperidine-1-carboxamide (2b:X═NH) is prepared by reacting 4-oxo-piperidine-1-carbonyl chloride.

Scheme 3 illustrates the general preparation of11-deoxo-11-hydroxyimino-3,4-spiro-piperidyl-rifamycins (3c). Thecompounds of (3c) are synthesized from the reaction of 11-oxy-rifamycincompound (2c) with hydroxylamine (or its HCl salt) at a temperaturerange from 10° C. to 70° C., in organic solvent, such as THF ormethanol, in the presence or absence of base, such as pyridine.

The above syntheses schemes are preferred schemes for the preparation ofthe indicated types of compounds. It is apparent to one skilled in artthat other sequences of the reactions, and alternative reagents can beused for the synthesis of the rifamycin derivatives of the presentdisclosure. These alternatives for the synthesis of the derivatives arewithin the scope of this invention.

The following examples provide synthesis schemes for specific rifabutinderivative compositions. All starting material used in these examplesare either purchased from commercial sources or prepared according topublished procedures. Reagents were purchased from commercial sourcesand used without further purification. Reactions with moisture-sensitivereagents were performed under a nitrogen atmosphere. Concentration ofsolutions was performed by reduced pressure (in vacuum) rotaryevaporation. Column flash chromatography was performed using silica gel60 as stationary phase. The preparative thin-layer chromatography (TLC)was performed using glass plates (20×20 cm) of silica gel (60 F254,thickness 1 mm or 2 mm).

Proton nuclear magnetic resonance (1H-NMR) spectra were recorded on aVarian Inova 300, or 500 MHz magnetic resonance spectrometer. 1H-NMRrefers to proton nuclear magnetic resonance spectroscopy with chemicalshifts reported in ppm (parts per million) downfield fromtetramethylsilane or referred to a residue signal of solvent(CHCl₃=7.27). 13C-NMR spectra were recorded on Varian Inova 500 MHzspectrometer operating at 125 MHz and Chemical shifts were reported inppm and referenced to residual solvent signals (CHCl₃=d 77.23 forcarbon)

The high resolution mass spectra (HRMS) were carried out in aBruker-micrOTOF-QII spectrometer, using electro spray ionizationpositive (ESI+) method and reported as M+H or M+Na, referring toprotonated molecular ion or its sodium complex.

The following examples are for illustration purposes and are notintended to limit the scope of the invention. It will be apparent to oneskilled in the art that the compounds of current invention can beprepared by a variety of synthetic routes, including but not limited tosubstitution of appropriate reagents, solvents or catalyst, change ofreaction sequence, and variation of protecting groups.

General Procedure (A) for Synthesis of Compounds (1c in Scheme 1):

In a sealed reaction tube, a reaction mixture of3-amino-4-imino-rifamycin S (1a) (0.1 mmol), piperidone or hexanon-typeof ketone (1b) (0.2-0.3 mmol), and ammonium acetate (1 mmol) in THF (3ml) was stirred at 60° C. overnight under nitrogen. The reaction mixturewas allowed to cool to room temperature and diluted with DCM (20 ml) andwater (20 ml). The aqueous phase was extracted with DCM (2×20 ml). Thecombined organic phase was washed with water (20 ml) and brine. Theorganic phase was dried over anhydrous sodium sulphate, filtered andconcentrated under vacuum. The residue was purified either by silica gelcolumn chromatography or by silica gel preparative thin-layerchromatography with methanol in DCM as eluent to give the product aspurple solid.

General Procedure (B) for Synthesis of Compounds (2c in Scheme 1):

In a round bottom flask with condenser, a reaction mixture of3-amino-4-imino-rifamycin S (1a) (0.1 mmol), piperidone or hexanon-typeof ketone (1b) (0.2-0.3 mmol), and ammonium acetate (0.2-0.3 mmol) inTHF (8 ml) was stirred at 75° C. overnight under nitrogen. The reactionmixture was allowed to cool to room temperature and diluted with DCM (20ml) and water (20 ml). The aqueous phase was extracted with DCM (2×20ml). The combined organic phase was washed with water (20 ml) and brine.The organic phase was dried over anhydrous sodium sulphate, filtered andconcentrated under vacuum. The residue was purified either by silica gelcolumn chromatography or by silica gel preparative thin-layerchromatography with methanol in DCM as eluent to give the product aspurple solid.

General Procedure (C) for Synthesis of Compounds (1d in Scheme 1 and 2din Scheme 2):

To a solution of rifamycin 11-imine or 11-oxo-compound (1c or 2c) (0.1mmol) in THF (4 ml) was added a suspension of NaBH4 (0.2 mmol) inethanol (4 ml) at room temperature. The reaction mixture stirred at roomtemperature for 1.5 hours and diluted with ethyl acetate (20 ml) andwater (20 ml). The aqueous phase was extracted with ethyl acetate (2×20ml). The combined organic phase was washed with water and brine. Theorganic phase was dried over anhydrous sodium sulphate, filtered andconcentrated under vacuum. The residue was purified either by silica gelcolumn chromatography or by silica gel preparative thin-layerchromatography with methanol in DCM as eluent to give the product aspurple solid.

Preparation of RTI-3311-deoxy-11-imino-4-deoxy-3,4[2-spiro-[1-(t-butyloxycarbonyl)-piperidin-4-yl]]-(1H)-imidazo-(2,5-dihydro)rifamycinS

Following the general procedure (A), the title compound was obtained asa pure solid. HRMS (ESI⁺): 890.4570 (M+H)⁺; calculated for (M+H)⁺:890.4553; 1H-NMR (300 MHz, CDCl₃) δ −0.09 (d, J=7 Hz, 3H), 0.61 (d, J=7Hz, 3H), 0.84 (d, J=7 Hz, 3H), 1.04 (d, J=7 Hz, 3H), 1.44 (m, 1H), 1.50(s, 9H), 1.6-1.85 (m, 4H), 1.88 (s, 3H), 1.9-2.15 (m, 2H), 2.02 (s, 3H),2.05 (s, 3H), 2.35 (s, 3H), 2.40 (m, 1H), 3.00 (m, 1H), 3.09 (s, 3H),3.33 (m, 1H), 3.49 (s, 1H), 3.60 (d, J=5 Hz, 1H), 3.68 (d, J=10 Hz, 1H),3.6-3.8 (br, 2H), 3.95-4.1 (br, 2H), 4.72 (d, J=10 Hz, 1H), 5.07 (dd,J=12 and 7 Hz, 1H), 6.03 (dd, J=16 and 7 Hz, 1H), 6.16 (d, J=12 Hz, 1H),6.28 (d, J=10 Hz, 1H), 6.40 (dd, J=16 and 10 Hz, 1H), 8.26 (s, 1H), 8.71(bs, 1H), 12.93 (s, 1H), 14.21 (s, 1H).

Preparation of RTI-3511-deoxy-11-imino-4-deoxy-3,4[2-spiro-tetrahydrothiopyran-4-yl]-(1H)-imidazo-(2,5-dihydro)rifamycinS

Following the general procedure (A), the title compound was obtained asa pure solid. HRMS (ESI⁺): 807.3665 (M+H)⁺; calculated for (M+H)⁺:807.3640; RTI-035A, 1H-NMR (300 MHz, CDCl3): −0.08 (d, J=7 Hz, 3H), 0.62(d, J=7 Hz, 3H), 0.85 (d, J=7 Hz, 3H), 1.05 (d, J=7 Hz, 3H), 1.44 (m,1H), 1.75-1.85 (m, 2H), 1.89 (s, 3H), 2.02 (s, 3H), 2.07 (s, 3H),1.9-2.15 (m, 4H), 2.35 (s, 3H), 2.40 (m, 1H), 2.75-2.9 (m, 2H), 3.00 (m,1H), 3.09 (s, 3H), 3.15-3.3 (m, 2H), 3.34 (dd, J=7 and 2 Hz, 1H), 3.47(s, 1H), 3.60 (d, J=6 Hz, 1H), 3.68 (d, J=10 Hz, 1H), 4.72 (d, J=10 Hz,1H), 5.07 (dd, J=12 and 8 Hz, 1H), 6.03 (dd, J=15 and 6 Hz, 1H), 6.18(d, J=12 Hz, 1H), 6.30 (d, J=10 Hz, 1H), 6.40 (dd, J=15 and 10 Hz, 1H),8.23 (s, 1H), 8.78 (s, 1H), 12.93 (s, 1H), 14.21 (s, 1H).

Preparation of RTI-4411-deoxy-11-imino-4-deoxy-3,4[2-spiro-[piperidin-4-yl]]-(1H)-imidazo-(2,5-dihydro)rifamycinS

Following the general procedure (A), the title compound was obtained asa pure solid. HRMS (ESI⁺): 790.4078 (M+H)⁺; calculated for (M+H)⁺:790.4029; RTI-044C, 1H-NMR (300 MHz, CDCl3): −0.08 (d, J=7 Hz, 3H), 0.61(d, J=7 Hz, 3H), 0.85 (d, J=7 Hz, 3H), 1.05 (d, J=7 Hz, 3H), 1.44 (m,1H), 1.75-1.85 (m, 2H), 1.89 (s, 3H), 2.02 (s, 3H), 2.07 (s, 3H),1.85-2.15 (m, 4H), 2.35 (s, 3H), 2.40 (m, 1H), 3.00 (m, 1H), 3.09 (s,3H), 3.15-3.3 (m, 2H), 3.3-3.45 (m, 4H), 3.50 (s, 1H), 3.45-3.65 (br,1H), 3.69 (d, J=10 Hz, 1H), 4.75 (d, J=10 Hz, 1H), 5.08 (dd, J=12 and 7Hz, 1H), 6.04 (dd, J=15 and 6 Hz, 1H), 6.18 (d, J=12 Hz, 1H), 6.30 (d,J=10 Hz, 1H), 6.42 (dd, J=15 and 10 Hz, 1H), 8.24 (s, 1H), 8.82 (s, 1H),13.00 (s, 1H), 14.28 (s, 1H).

Preparation of RTI-4611-deoxy-11-imino-4-deoxy-3,4[2-spiro-cyclohexyl]-(1H)-imidazo-(2,5-dihydro)rifamycinS

Following the general procedure (A), the title compound was obtained asa pure solid. HRMS (ESI⁺): 789.4122 (M+H)⁺; calculated for (M+H)⁺:789.4076; RTI-046C, 1H-NMR (300 MHz, CDCl3): −0.09 (d, J=7 Hz, 3H), 0.61(d, J=7 Hz, 3H), 0.85 (d, J=7 Hz, 3H), 1.04 (d, J=7 Hz, 3H), 1.44 (m,1H), 1.7-1.9 (m, 10H), 1.89 (s, 3H), 2.01 (s, 3H), 2.06 (s, 3H),1.95-2.1 (m, 2H), 2.33 (s, 3H), 2.40 (m, 1H), 3.00 (m, 1H), 3.08 (s,3H), 3.34 (dd, J=7 and 3 Hz, 1H), 3.45 (s, 1H), 3.62 (d, J=6 Hz, 1H),3.68 (d, J=10 Hz, 1H), 4.75 (d, J=10 Hz, 1H), 5.08 (dd, J=12 and 7 Hz,1H), 6.03 (dd, J=15 and 6 Hz, 1H), 6.16 (d, J=12 Hz, 1H), 6.27 (d, J=10Hz, 1H), 6.40 (dd, J=15 and 10 Hz, 1H), 8.21 (s, 1H), 8.87 (s, 1H),13.00 (s, 1H), 14.33 (s, 1H).

Preparation of RTI-4911-deoxy-11-imino-4-deoxy-3,4[2-spiro-[1-(benzyl)-piperidin-4-yl]]-(1H)-imidazo-(2,5-dihydro)rifamycinS

Following the general procedure (A), the title compound was obtained asa pure solid. HRMS (ESI⁺): 880.4535 (M+H)⁺; calculated for (M+H)⁺:880.4498; RTI-049A, 1H-NMR (300 MHz, CDCl3): −0.09 (d, J=7 Hz, 3H), 0.60(d, J=7 Hz, 3H), 0.84 (d, J=7 Hz, 3H), 1.04 (d, J=7 Hz, 3H), 1.44 (m,1H), 1.65-1.85 (m, 2H), 1.91 (s, 3H), 2.01 (s, 3H), 2.07 (s, 3H), 2.35(s, 3H), 2.40 (m, 1H), 2.47 (t, J=6 Hz, 2H), 2.76 (t, J=6 Hz, 2H),2.8-2.95 (m, 4H), 3.00 (m, 1H), 3.09 (s, 3H), 3.33 (dd, J=7 and 2 Hz,1H), 3.46 (s, 1H), 3.60-3.72 (m, 4H), 4.74 (d, J=10 Hz, 1H), 5.08 (dd,J=12 and 7 Hz, 1H), 6.04 (dd, J=16 and 7 Hz, 1H), 6.18 (d, J=12 Hz, 1H),6.27 (d, J=10 Hz, 1H), 6.40 (dd, J=16 and 10 Hz, 1H), 7.3-7.45 (m, 5H),8.22 (s, 1H), 8.80 (s, 1H), 12.99 (s, 1H), 14.31 (s, 1H).

Preparation of RTI-5111-deoxy-11-imino-4-deoxy-3,4[2-spiro-[1-(2-methoxyethyl)-piperidin-4-yl]]-(1H)-imidazo-(2,5-dihydro)rifamycinS

Following the general procedure (A), the title compound was obtained asa pure solid. HRMS (ESI⁺): 848.4487 (M+H)⁺; calculated for (M+H)⁺:848.4447; RTI-051A, 1H-NMR (300 MHz, CDCl3): −0.09 (d, J=7 Hz, 3H), 0.61(d, J=7 Hz, 3H), 0.85 (d, J=7 Hz, 3H), 1.04 (d, J=7 Hz, 3H), 1.44 (m,1H), 1.65-1.85 (m, 4H), 1.90 (s, 3H), 2.02 (s, 3H), 2.07 (s, 3H),1.85-2.15 (br, 2H), 2.35 (s, 3H), 2.40 (m, 1H), 2.79 (t, J=5 Hz, 2H),2.85-2.95 (m, 4H), 3.00 (m, 1H), 3.09 (s, 3H), 3.33 (dd, J=7 and 2 Hz,1H), 3.41 (s, 3H), 3.49 (s, 1H), 3.59 (t, J=5 Hz, 2H), 3.64 (d, J=6 Hz,1H), 3.68 (d, J=10 Hz, 1H), 4.75 (d, J=10 Hz, 1H), 5.08 (dd, J=12 and 7Hz, 1H), 6.04 (dd, J=15 and 7 Hz, 1H), 6.16 (d, J=12 Hz, 1H), 6.27 (d,J=10 Hz, 1H), 6.41 (dd, J=15 and 10 Hz, 1H), 8.25 (s, 1H), 8.77 (s, 1H),12.94 (s, 1H), 14.31 (s, 1H).

Preparation of RTI-5311-deoxy-11-imino-4-deoxy-3,4[2-spiro-[1-(2-morpholinoethyl)-piperidin-4-yl]]-(1H)-imidazo-(2,5-dihydro)rifamycinS

Following the general procedure (A), the title compound was obtained asa pure solid. HRMS (ESI⁺): 903.4904 (M+H)⁺; calculated for (M+H)⁺:903.4869; RTI-053A, 1H-NMR (300 MHz, CDCl3): −0.09 (d, J=7 Hz, 3H), 0.61(d, J=7 Hz, 3H), 0.85 (d, J=7 Hz, 3H), 1.04 (d, J=7 Hz, 3H), 1.44 (m,1H), 1.65-1.85 (m, 4H), 1.90 (s, 3H), 2.02 (s, 3H), 2.07 (s, 3H),1.85-2.15 (br, 2H), 2.34 (s, 3H), 2.40 (m, 1H), 2.5-2.65 (m, 6H), 2.74(m, 2H), 2.85-2.95 (m, 4H), 3.00 (m, 1H), 3.09 (s, 3H), 3.33 (dd, J=7and 2 Hz, 1H), 3.49 (s, 1H), 3.64 (d, J=6 Hz, 1H), 3.68 (d, J=10 Hz,1H), 3.74 (t, J=5 Hz, 4H), 4.75 (d, J=10 Hz, 1H), 5.08 (dd, J=12 and 7Hz, 1H), 6.04 (dd, J=15 and 7 Hz, 1H), 6.16 (d, J=12 Hz, 1H), 6.28 (d,J=10 Hz, 1H), 6.40 (dd, J=15 and 10 Hz, 1H), 8.25 (s, 1H), 8.77 (s, 1H),12.94 (s, 1H), 14.29 (s, 1H).

Preparation of RTI-5711-deoxy-11-imino-4-deoxy-3,4[2-spiro-[1-(cyclobutylmethyl)-piperidin-4-yl]]-(1H)-imidazo-(2,5-dihydro)rifamycinS

Following the general procedure (A), the title compound was obtained asa pure solid. HRMS (ESI⁺): 858.4690 (M+H)⁺; calculated for (M+H)⁺:858.4655; RTI-057A, 1H-NMR (300 MHz, CDCl3): −0.09 (d, J=7 Hz, 3H), 0.61(d, J=7 Hz, 3H), 0.85 (d, J=7 Hz, 3H), 1.04 (d, J=7 Hz, 3H), 1.44 (m,1H), 1.7-1.85 (m, 8H), 1.90 (s, 3H), 1.9-2.15 (m, 4H), 2.02 (s, 3H),2.07 (s, 3H), 2.35 (s, 3H), 2.40 (m, 1H), 2.60 (m, 3H), 2.7-2.9 (br,4H), 3.00 (m, 1H), 3.09 (s, 3H), 3.34 (dd, J=7 and 2 Hz, 1H), 3.46 (s,1H), 3.63 (d, J=6 Hz, 1H), 3.68 (d, J=10 Hz, 1H), 4.75 (d, J=10 Hz, 1H),5.08 (dd, J=12 and 7 Hz, 1H), 6.03 (dd, J=16 and 7 Hz, 1H), 6.17 (d,J=12 Hz, 1H), 6.28 (d, J=10 Hz, 1H), 6.40 (dd, J=16 and 10 Hz, 1H), 8.22(s, 1H), 8.80 (s, 1H), 12.95 (s, 1H), 14.31 (s, 1H).

Preparation of RTI-5911-deoxy-11-imino-4-deoxy-3,4[2-spiro-[1-(cclopropvlmethyl)-piperidin-4-yl]]-(1H)-imidazo-(2,5-dihydro)rifamycinS

Following the general procedure (A), the title compound was obtained asa pure solid. HRMS (ESI⁺): 844.4536 (M+H)⁺; calculated for (M+H)⁺:844.4498; RTI-059A, 1H-NMR (300 MHz, CDCl3): −0.09 (d, J=7 Hz, 3H), 0.18(m, 2H), 0.57 (m, 2H), 0.61 (d, J=7 Hz, 3H), 0.84 (d, J=7 Hz, 3H), 0.93(m, 1H), 1.04 (d, J=7 Hz, 3H), 1.44 (m, 1H), 1.7-1.85 (m, 4H), 1.90 (s,3H), 1.95-2.15 (br, 2H), 2.02 (s, 3H), 2.07 (s, 3H), 2.35 (s, 3H), 2.40(m, 1H), 2.46 (d, J=7 Hz, 2H), 2.8-3.05 (m, 5H), 3.09 (s, 3H), 3.35 (dd,J=7 and 2 Hz, 1H), 3.49 (s, 1H), 3.63 (d, J=6 Hz, 1H), 3.68 (d, J=10 Hz,1H), 4.74 (d, J=10 Hz, 1H), 5.07 (dd, J=12 and 7 Hz, 1H), 6.03 (dd, J=16and 7 Hz, 1H), 6.17 (d, J=12 Hz, 1H), 6.28 (d, J=10 Hz, 1H), 6.40 (dd,J=16 and 10 Hz, 1H), 8.25 (s, 1H), 8.78 (s, 1H), 12.93 (s, 1H), 14.31(s, 1H).

Preparation of RTI-6011-deoxy-11-imino-4-deoxy-3,4[2-spiro-[1-(isopropyl)-piperidin-4-yl]]-(1H)-imidazo-(2,5-dihydro)rifamycinS

Following the general procedure (A), the title compound was obtained asa pure solid. HRMS (ESI⁺): 832.4542 (M+H)⁺; calculated for (M+H)⁺:832.4498; RTI-060A, 1H-NMR (300 MHz, CDCl3): −0.09 (d, J=7 Hz, 3H), 0.60(d, J=7 Hz, 3H), 0.84 (d, J=7 Hz, 3H), 1.04 (d, J=7 Hz, 3H), 1.16 (d,J=6 Hz, 6H), 1.44 (m, 1H), 1.7-1.8 (m, 4H), 1.88 (s, 3H), 1.95-2.15 (br,2H), 2.01 (s, 3H), 2.05 (s, 3H), 2.33 (s, 3H), 2.40 (m, 1H), 2.75-3.05(m, 6H), 3.08 (s, 3H), 3.34 (dd, J=7 and 2 Hz, 1H), 3.47 (s, 1H), 3.64(d, J=6 Hz, 1H), 3.68 (d, J=10 Hz, 1H), 4.75 (d, J=10 Hz, 1H), 5.07 (dd,J=12 and 7 Hz, 1H), 6.03 (dd, J=16 and 7 Hz, 1H), 6.16 (d, J=12 Hz, 1H),6.27 (d, J=10 Hz, 1H), 6.40 (dd, J=16 and 10 Hz, 1H), 8.22 (s, 1H), 8.76(s, 1H), 12.91 (s, 1H), 14.31 (s, 1H).

Preparation of RTI-6111-deoxy-11-imino-4-deoxy-3,4[2-spiro-[1-(t-ethyloxycarbonyl)-piperidin-4-yl]]-(1H)-imidazo-(2,5-dihydro)rifamycinS

Following the general procedure (A), the title compound was obtained asa pure solid. HRMS (ESI⁺): 862.4270 (M+H)⁺; calculated for (M+H)⁺:862.4240: RTI-61A, 1H-NMR (300 MHz, CDCl3): −0.08 (d, J=7 Hz, 3H), 0.62(d, J=7 Hz, 3H), 0.84 (d, J=7 Hz, 3H), 1.04 (d, J=7 Hz, 3H), 1.30 (t,J=7 Hz, 3H), 1.44 (m, 1H), 1.6-1.85 (m, 4H), 1.89 (s, 3H), 2.0-2.15 (m,2H), 2.02 (s, 3H), 2.06 (s, 3H), 2.35 (s, 3H), 2.40 (m, 1H), 3.00 (m,1H), 3.09 (s, 3H), 3.33 (m, 1H), 3.50 (s, 1H), 3.61 (d, J=5 Hz, 1H),3.68 (d, J=10 Hz, 1H), 3.6-3.8 (br, 2H), 4.0-4.2 (br, 2H), 4.21 (q, J=7Hz, 2H), 4.72 (d, J=10 Hz, 1H), 5.07 (dd, J=12 and 7 Hz, 1H), 6.03 (dd,J=16 and 7 Hz, 1H), 6.17 (d, J=12 Hz, 1H), 6.29 (d, J=10 Hz, 1H), 6.41(dd, J=16 and 10 Hz, 1H), 8.26 (s, 1H), 8.72 (bs, 1H), 12.93 (s, 1H),14.21 (s, 1H).

Preparation of RTI-6311-deoxy-11-imino-4-deoxy-3,4[2-spiro-[1-(acetyl)-piperidin-4-yl]]-(1H)-imidazo-(2,5-dihydro)rifamycinS

Following the general procedure (A), the title compound was obtained asa pure solid. HRMS (ESI⁺): 832.4181 (M+H)⁺; calculated for (M+H)⁺:832.4134. RTI-63A, 1H-NMR (300 MHz, CDCl3): −0.06 (d, J=7 Hz, 3H), 0.62(d, J=7 Hz, 3H), 0.85 (d, J=7 Hz, 3H), 1.04 (d, J=7 Hz, 3H), 1.45 (m,1H), 1.6-1.85 (m, 4H), 1.89 (s, 3H), 2.03 (s, 3H), 2.06 (s, 3H), 2.0-2.2(m, 2H), 2.20 (s, 3H), 2.35 (s, 3H), 2.40 (m, 1H), 3.00 (m, 1H), 3.10(s, 3H), 3.33 (m, 1H), 3.47 (s, 0.4H), 3.51 (s, 0.6H), 3.55-3.70 (m,3H), 3.90 (m, 2H), 4.48 (m, 1H), 4.73 (m, 1H), 5.07 (m, 1H), 6.03 (dd,J=16 and 6 Hz, 1H), 6.18 (d, J=12 Hz, 1H), 6.29 (d, J=10 Hz, 1H), 6.38(m, 1H), 8.25 (s, 1H), 8.66 (s, 0.6H), 8.71 (s, 0.4H), 12.92 (s, 1H),14.16 (s, 0.4H), 14.19 (s, 0.6H).

Preparation of RTI-6411-deoxy-11-imino-4-deoxy-3,4[2-spiro-[1-(n-propyl)-piperidin-4-yl]]-(1H)-imidazo-(2,5-dihydro)rifamycinS

Following the general procedure (A), the title compound was obtained asa pure solid. HRMS (ESI⁺): 832.4552 (M+H)⁺; calculated for (M+H)⁺:832.4498; RTI-064A, 1H-NMR (300 MHz, CDCl3): −0.09 (d, J=7 Hz, 3H), 0.61(d, J=7 Hz, 3H), 0.85 (d, J=7 Hz, 3H), 0.96 (t, J=7 Hz, 3H), 1.04 (d,J=7 Hz, 3H), 1.44 (m, 1H), 1.55-1.65 (m, 2H), 1.7-1.85 (m, 4H), 1.90 (s,3H), 1.95-2.15 (br, 2H), 2.02 (s, 3H), 2.07 (s, 3H), 2.35 (s, 3H), 2.40(m, 1H), 2.54 (m, 2H), 2.8-2.9 (m, 4H), 3.00 (m, 1H), 3.09 (s, 3H), 3.35(dd, J=7 and 2 Hz, 1H), 3.46 (s, 1H), 3.62 (d, J=6 Hz, 1H), 3.67 (d,J=10 Hz, 1H), 4.75 (d, J=10 Hz, 1H), 5.07 (dd, J=12 and 7 Hz, 1H), 6.03(dd, J=16 and 7 Hz, 1H), 6.17 (d, J=12 Hz, 1H), 6.27 (d, J=10 Hz, 1H),6.40 (dd, J=16 and 10 Hz, 1H), 8.21 (s, 1H), 8.78 (s, 1H), 12.95 (s,1H), 14.30 (s, 1H).

Preparation of RTI-6511-deox-11-imino-4-deoxy-3,4[2-spiro-[1-(cyclopropyl)-piperidin-4-yl]]-(1H)-imidazo-(2,5-dihydro)rifamycinS

Following the general procedure (A), the title compound was obtained asa pure solid. HRMS (ESI⁺): 830.4386 (M+H)⁺; calculated for (M+H)⁺:830.4342; RTI-065A. 1H-NMR (300 MHz, CDCl3): −0.09 (d, J=7 Hz, 3H),0.45-0.55 (m, 5H), 0.61 (d, J=7 Hz, 3H), 0.85 (d, J=7 Hz, 3H), 1.04 (d,J=7 Hz, 3H), 1.44 (m, 1H), 1.7-1.85 (m, 4H), 1.90 (s, 3H), 1.95-2.15(br, 2H), 2.02 (s, 3H), 2.07 (s, 3H), 2.35 (s, 3H), 2.40 (m, 1H),2.9-3.1 (m, 5H), 3.09 (s, 3H), 3.35 (dd, J=7 and 2 Hz, 1H), 3.46 (s,1H), 3.63 (d, J=6 Hz, 1H), 3.67 (d, J=10 Hz, 1H), 4.75 (d, J=10 Hz, 1H),5.08 (dd, J=12 and 7 Hz, 1H), 6.04 (dd, J=16 and 7 Hz, 1H), 6.17 (d,J=12 Hz, 1H), 6.27 (d, J=10 Hz, 1H), 6.40 (dd, J=16 and 10 Hz, 1H), 8.21(s, 1H), 8.79 (s, 1H), 12.97 (s, 1H), 14.30 (s, 1H).

Preparation of RTI-6611-deox-11-imino-4-deoxy-3,4[2-spiro-[1-(ethyl)-piperidin-4-yl]]-(1H)-imidazo-(2,5-dihydro)rifamycinS

Following the general procedure (A), the title compound was obtained asa pure solid. HRMS (ESI⁺): 818.4388 (M+H)⁺; calculated for (M+H)⁺:818.4342; RTI-066A, 1H-NMR (300 MHz, CDCl3): −0.08 (d, J=7 Hz, 3H), 0.61(d, J=7 Hz, 3H), 0.85 (d, J=7 Hz, 3H), 1.04 (d, J=7 Hz, 3H), 1.18 (t,J=7 Hz, 3H), 1.44 (m, 1H), 1.7-1.85 (m, 4H), 1.90 (s, 3H), 1.95-2.15(br, 2H), 2.02 (s, 3H), 2.07 (s, 3H), 2.35 (s, 3H), 2.40 (m, 1H), 2.64(q, J=7 Hz, 2H), 2.8-2.95 (m, 4H), 3.00 (m, 1H), 3.09 (s, 3H), 3.35 (d,J=7 Hz, 1H), 3.46 (s, 1H), 3.63 (d, J=6 Hz, 1H), 3.67 (d, J=10 Hz, 1H),4.75 (d, J=10 Hz, 1H), 5.08 (dd, J=12 and 7 Hz, 1H), 6.04 (dd, J=16 and7 Hz, 1H), 6.16 (d, J=12 Hz, 1H), 6.27 (d, J=10 Hz, 1H), 6.40 (dd, J=16and 10 Hz, 1H), 8.22 (s, 1H), 8.77 (s, 1H), 12.95 (s, 1H), 14.29 (s,1H).

Preparation of RTI-6711-deoxy-11-imino-4-deoxy-3,4[2-spiro-[1-(beRTIoyl)-piperidin-4-yl]]-(1H)-imidazo-(2,5-dihydro)rifamycinS

Following the general procedure (A), the title compound was obtained asa pure solid. HRMS (ESI⁺): 916.4169 (M+Na)⁺; calculated for (M+Na)⁺:916.4109. RTI-67A, 1H-NMR (300 MHz, CDCl3): −0.07 (br, 3H), 0.60 (br,3H), 0.84 (br, 3H), 1.02 (d, J=7 Hz, 3H), 1.45 (m, 1H), 1.6-1.85 (m,4H), 1.88 (s, 3H), 2.00 (s, 3H), 2.04 (s, 3H), 1.9-2.2 (m, 2H), 2.34 (s,3H), 2.40 (m, 1H), 3.00 (m, 1H), 3.08 (s, 3H), 3.2-3.9 (br, 7H), 4.2(br, 1H), 4.6 (br, 1H), 5.05 (br, 1H), 6.0 (br, 1H), 6.18 (br, 1H), 6.29(br, 1H), 6.40 (br, 1H), 7.40 (m, 2H), 7.45 (m, 3H), 8.25 (s, 1H), 8.6(brs, 1H), 12.93 (s, 1H), 14.16 (s, 1H).

Preparation of RTI-6811-deoxy-11-imino-4-deoxy-3,4[2-spiro-[1-(benzyloxycarbonyl)-piperidin-4-yl]]-(1H)-imidazo-(2,5-dihydro)rifamycinS

Following the general procedure (A), the title compound was obtained asa pure solid. HRMS (ESI⁺): 924.4435 (M+H)⁺; calculated for (M+H):924.4396; RTI-68A, 1H-NMR (300 MHz, CDCl3): −0.09 (d, J=7 Hz, 3H), 0.60(d, J=7 Hz, 3H), 0.84 (d, J=7 Hz, 3H), 1.04 (d, J=7 Hz, 3H), 1.44 (m,1H), 1.6-1.85 (m, 4H), 1.88 (s, 3H), 2.0-2.15 (m, 2H), 2.02 (s, 3H),2.05 (s, 3H), 2.35 (s, 3H), 2.40 (m, 1H), 3.00 (m, 1H), 3.09 (s, 3H),3.33 (br, 1H), 3.49 (s, 1H), 3.60 (d, J=5 Hz, 1H), 3.68 (d, J=10 Hz,1H), 3.6-3.8 (br, 2H), 4.0-4.2 (m, 2H), 4.72 (d, J=10 Hz, 1H), 5.07 (dd,J=12 and 7 Hz, 1H), 5.20 (s, 2H), 6.03 (dd, J=16 and 7 Hz, 1H), 6.17 (d,J=12 Hz, 1H), 6.29 (d, J=10 Hz, 1H), 6.41 (dd, J=16 and 10 Hz, 1H), 7.38(m, 5H), 8.26 (s, 1H), 8.70 (bs, 1H), 12.92 (s, 1H), 14.20 (s, 1H).

Preparation of RTI-6911-deoxy-11-imino-4-deoxy-3,4[2-spiro-[1-(methyl)-piperidin-4-yl]]-(1H)-imidazo-(2,5-dihydro)rifamycinS

Following the general procedure (A), the title compound was obtained asa pure solid. HRMS (ESI⁺): 804.4213 (M+H)⁺; calculated for (M+H)⁺:804.4185: RTI-069A, 1H-NMR (300 MHz, CDCl3): −0.08 (d, J=7 Hz, 3H), 0.61(d, J=7 Hz, 3H), 0.85 (d, J=7 Hz, 3H), 1.04 (d, J=7 Hz, 3H), 1.45 (m,1H), 1.7-1.85 (m, 4H), 1.90 (s, 3H), 1.95-2.15 (br, 2H), 2.02 (s, 3H),2.07 (s, 3H), 2.35 (s, 3H), 2.40 (m, 1H), 2.49 (s, 3H), 2.7-2.95 (m,4H), 3.00 (m, 1H), 3.09 (s, 3H), 3.34 (d, J=7 Hz, 1H), 3.48 (s, 1H),3.63 (d, J=6 Hz, 1H), 3.68 (d, J=10 Hz, 1H), 4.75 (d, J=10 Hz, 1H), 5.08(dd, J=12 and 7 Hz, 1H), 6.04 (dd, J=16 and 7 Hz, 1H), 6.17 (d, J=12 Hz,1H), 6.27 (d, J=10 Hz, 1H), 6.40 (dd, J=16 and 10 Hz, 1H), 8.23 (s, 1H),8.77 (s, 1H), 12.95 (s, 1H), 14.29 (s, 1H).

Preparation of RTI-7011-deoxy-11-imino-4-deoxy-3,4[2-spiro-[1-(2-methylpropyl)-piperidin-4-yl]]-(1H)-imidazo-(2,5-dihydro)rifamycinS

Following the general procedure (A), the title compound was obtained asa pure solid. HRMS (ESI⁺): 846.4682 (M+H)⁺; calculated for (M+H)⁺:846.4655; RTI-070A, 1H-NMR (500 MHz, CDCl3): −0.09 (d, J=7 Hz, 3H), 0.60(d, J=7 Hz, 3H), 0.84 (d, J=7 Hz, 3H), 0.94 (d, J=7 Hz, 6H), 1.04 (d,J=7 Hz, 3H), 1.44 (m, 1H), 1.74-1.85 (m, 3H), 1.89 (s, 3H), 1.9-2.15 (m,4H), 2.01 (s, 3H), 2.05 (s, 3H), 2.29 (d, J=7 Hz, 2H), 2.33 (s, 3H),2.40 (m, 1H), 2.75-2.85 (m, 4H), 3.00 (m, 1H), 3.08 (s, 3H), 3.33 (dd,J=7 and 2 Hz, 1H), 3.46 (s, 1H), 3.63 (d, J=6 Hz, 1H), 3.68 (d, J=10 Hz,1H), 4.75 (dd, J=10 and 2 Hz, 1H), 5.07 (dd, J=12 and 7 Hz, 1H), 6.03(dd, J=16 and 7 Hz, 1H), 6.16 (d, J=12 Hz, 1H), 6.27 (d, J=10 Hz, 1H),6.40 (dd, J=16 and 10 Hz, 1H), 8.23 (s, 1H), 8.78 (s, 1H), 12.96 (s,1H), 14.30 (s, 1H), 13C-NMR (125 MHz, CDCl3).

Preparation of RTI-7411-deoxy-11-imino-4-deoxy-3,4[2-spiro-[1-(phenylaminocarbonyl)-piperidin-4-yl]]-(1H)-imidazo-(2,5-dihydro)rifamycinS

Following the general procedure (A), the title compound was obtained asa pure solid. HRMS (ESI⁺): 909.4433 (M+H)⁺; calculated for (M+H)⁺:909.4400; RTI-074A, 1H-NMR (300 MHz, CDCl3): −0.07 (d, J=7 Hz, 3H), 0.62(d, J=7 Hz, 3H), 0.84 (d, J=7 Hz, 3H), 1.04 (d, J=7 Hz, 3H), 1.44 (m,1H), 1.6-1.85 (m, 3H), 1.89 (s, 3H), 1.9-2.25 (m, 3H), 2.02 (s, 3H),2.05 (s, 3H), 2.35 (s, 3H), 2.40 (m, 1H), 3.00 (m, 1H), 3.09 (s, 3H),3.33 (m, 1H), 3.51 (s, 1H), 3.61 (d, J=6 Hz, 1H), 3.68 (d, J=10 Hz, 1H),3.6-3.8 (br, 2H), 4.0-4.2 (br, 2H), 4.72 (d, J=10 Hz, 1H), 5.07 (dd,J=12 and 7 Hz, 1H), 6.03 (dd, J=16 and 7 Hz, 1H), 6.16 (d, J=12 Hz, 1H),6.28 (d, J=10 Hz, 1H), 6.40 (m, 2H), 7.15 (m, 1H), 7.34 (m, 4H), 8.27(s, 1H), 8.69 (s, 1H), 12.92 (s, 1H), 14.19 (s, 1H).

Preparation of RTI-7711-deoxy-11-imino-4-deox-3,4[2-spiro-[1-(ethyloxycarbonyl)-piperidin-4-yl]]-(1H)-imidazo-(2,5-dihydro)rifamycinS

Following the general procedure (A), the title compound was obtained asa pure solid. HRMS (ESI⁺): 876.4417 (M+H)⁺; calculated for (M+H)⁺:876.4396; RTI-77A, 1H-NMR (300 MHz, CDCl3): −0.08 (d, J=7 Hz, 3H), 0.61(d, J=7 Hz, 3H), 0.84 (d, J=7 Hz, 3H), 1.04 (d, J=7 Hz, 3H), 0.99 (t,J=7 Hz, 3H), 1.44 (m, 1H), 1.6-1.85 (m, 6H), 1.88 (s, 3H), 2.0-2.15 (m,2H), 2.02 (s, 3H), 2.05 (s, 3H), 2.35 (s, 3H), 2.40 (m, 1H), 3.00 (m,1H), 3.09 (s, 3H), 3.33 (m, 1H), 3.49 (s, 1H), 3.60 (d, J=5 Hz, 1H),3.68 (d, J=10 Hz, 1H), 3.6-3.8 (br, 2H), 4.0-4.2 (m, 4H), 4.72 (d, J=10Hz, 1H), 5.07 (dd, J=12 and 7 Hz, 1H), 6.03 (dd, J=16 and 7 Hz, 1H),6.17 (d, J=12 Hz, 1H), 6.28 (d, J=10 Hz, 1H), 6.41 (dd, J=16 and 10 Hz,1H), 8.25 (s, 1H), 8.7 (bs, 1H), 12.93 (s, 1H), 14.20 (s, 1H).

Preparation of RTI-8111-deoxy-11-imino-4-deoxy-3,4[2-spiro-[1-(isobutyloxycarbonyl)-piperidin-4-yl]]-(1H)-imidazo-(2,5-dihydro)rifamycinS

Following the general procedure (A), the title compound was obtained asa pure solid. HRMS (ESI⁺): 890.4552 (M+H)⁺; calculated for (M+H)⁺:890.4553; RTI-081, 1H-NMR (300 MHz, CDCl3): −0.09 (d, J=7 Hz, 3H), 0.61(d, J=7 Hz, 3H), 0.84 (d, J=7 Hz, 3H), 0.98 (d, J=7 Hz, 6H), 1.04 (d,J=7 Hz, 3H), 1.44 (m, 1H), 1.6-1.85 (m, 4H), 1.88 (s, 3H), 1.9-2.15 (m,3H), 2.01 (s, 3H), 2.05 (s, 3H), 2.35 (s, 3H), 2.40 (m, 1H), 3.00 (m,1H), 3.09 (s, 3H), 3.33 (m, 1H), 3.49 (s, 1H), 3.60 (d, J=6 Hz, 1H),3.68 (d, J=10 Hz, 1H), 3.6-3.8 (br, 2H), 3.95 (m, 2H), 4.0-4.2 (br, 2H),4.72 (d, J=10 Hz, 1H), 5.07 (dd, J=12 and 7 Hz, 1H), 6.03 (dd, J=16 and7 Hz, 1H), 6.16 (d, J=12 Hz, 1H), 6.27 (d, J=10 Hz, 1H), 6.40 (dd, J=16and 10 Hz, 1H), 8.25 (s, 1H), 8.7 (bs, 1H), 12.93 (s, 1H), 14.20 (s,1H).

Preparation ofRTI-8211-deoxy-11-imino-4-deoxy-3,4[2-spiro-[1-(ethylaminocarbonyl)-piperidin-4-yl]]-(1H)-imidazo-(2,5-dihydro)rifamycinS

Following the general procedure (A), the title compound was obtained asa pure solid. HRMS (ESI⁺): 883.4175 (M+Na)⁺; calculated for (M+Na)⁺:883.4218; RTI-082A, 1H-NMR (300 MHz, CDCl3): −0.08 (d, J=7 Hz, 3H), 0.61(d, J=7 Hz, 3H), 0.84 (d, J=7 Hz, 3H), 1.04 (d, J=7 Hz, 3H), 1.20 (t,J=7 Hz, 3H), 1.44 (m, 1H), 1.6-1.85 (m, 3H), 1.88 (s, 3H), 1.9-2.25 (m,3H), 2.02 (s, 3H), 2.05 (s, 3H), 2.35 (s, 3H), 2.40 (m, 1H), 3.00 (m,1H), 3.09 (s, 3H), 3.3-3.4 (m, 3H), 3.50 (s, 1H), 3.61 (d, J=6 Hz, 1H),3.68 (d, J=10 Hz, 1H), 3.6-3.7 (br, 2H), 3.8-4.0 (br, 2H), 4.52 (m, 1H),4.72 (d, J=10 Hz, 1H), 5.07 (dd, J=12 and 7 Hz, 1H), 6.03 (dd, J=16 and7 Hz, 1H), 6.16 (d, J=12 Hz, 1H), 6.28 (d, J=10 Hz, 1H), 6.40 (m, 1H),8.25 (s, 1H), 8.69 (s, 1H), 12.92 (s, 1H), 14.20 (s, 1H).

Preparation of RTI-834-deoxy-3,4[2-spiro-[1-(ethylaminocarbonyl)-piperidin-4-yl]]-(1H)-imidazo-(2,5-dihiydro)rifamycinS

Following the general procedure (B), the title compound was obtained asa pure solid. HRMS (ESI⁺): 884.4048 (M+Na)⁺; calculated for (M+Na)⁺:884.4058; RTI-083A, 1H-NMR (300 MHz, CDCl3): −0.04 (d, J=7 Hz, 3H), 0.61(d, J=7 Hz, 3H), 0.84 (d, J=7 Hz, 3H), 1.04 (d, J=7 Hz, 3H), 1.20 (t,J=7 Hz, 3H), 1.4-1.6 (m, 2H), 1.65-1.85 (m, 3H), 1.74 (s, 3H), 1.95-2.2(m, 2H), 2.02 (s, 3H), 2.04 (s, 3H), 2.35 (s, 3H), 2.40 (m, 1H), 3.00(m, 1H), 3.09 (s, 3H), 3.3-3.4 (m, 3H), 3.43 (s, 1H), 3.56 (d, J=6 Hz,1H), 3.68 (d, J=10 Hz, 1H), 3.7-4.0 (m, 4H), 4.50 (m, 1H), 4.72 (d, J=10Hz, 1H), 5.13 (dd, J=12 and 7 Hz, 1H), 6.03 (dd, J=16 and 7 Hz, 1H),6.18 (d, J=12 Hz, 1H), 6.27 (d, J=10 Hz, 1H), 6.38 (m, 1H), 8.18 (s,1H), 8.90 (s, 1H), 14.57 (s, 1H).

Preparation of RTI-8411-deoxy-11-imino-4-deoxy-3,4[2-spiro-[1-(isopropyloxycarbonyl)-piperidin-4-yl]]-(1H)-imidazo-(2,5-dihydro)rifamycinS

Following the general procedure (A), the title compound was obtained asa pure solid. HRMS (ESI⁺): 898.4203 (M+Na)⁺; calculated for (M+Na)⁺:898.4215; RTI-084A, 1H-NMR (300 MHz, CDCl3): −0.09 (d, J=7 Hz, 3H), 0.61(d, J=7 Hz, 3H), 0.84 (d, J=7 Hz, 3H), 1.04 (d, J=7 Hz, 3H), 1.30 (d,J=6 Hz, 6H), 1.44 (m, 1H), 1.6-1.85 (m, 4H), 1.88 (s, 3H), 1.9-2.15 (m,2H), 2.02 (s, 3H), 2.05 (s, 3H), 2.35 (s, 3H), 2.40 (m, 1H), 3.00 (m,1H), 3.09 (s, 3H), 3.33 (m, 1H), 3.50 (s, 1H), 3.61 (d, J=6 Hz, 1H),3.68 (d, J=10 Hz, 1H), 3.6-3.8 (br, 2H), 4.0-4.2 (br, 2H), 4.72 (d, J=10Hz, 1H), 4.99 (m, 1H), 5.07 (dd, J=12 and 7 Hz, 1H), 6.03 (dd, J=16 and7 Hz, 1H), 6.16 (d, J=12 Hz, 1H), 6.28 (d, J=10 Hz, 1H), 6.40 (dd, J=16and 10 Hz, 1H), 8.27 (s, 1H), 8.7 (bs, 1H), 12.93 (s, 1H), 14.21 (s,1H).

Preparation of RTI-864-deoxy-3,4[2-spiro-[1-(phenylaminocarlonyl)-piperidin-4-yl]]-(1H)-imidazo-(2,5-dihydro)rifamycinS

Following the general procedure (B), the title compound was obtained asa pure solid. HRMS (ESI⁺): 932.4038 (M+Na)⁺; calculated for (M+Na)⁺:932.4058; RTI-086A, 1H-NMR (300 MHz, CDCl3): −0.02 (d, J=7 Hz, 3H), 0.62(d, J=7 Hz, 3H), 0.84 (d, J=7 Hz, 3H), 1.04 (d, J=7 Hz, 3H), 1.4-1.6 (m,2H), 1.65-1.85 (m, 3H), 1.75 (s, 3H), 1.95-2.2 (m, 3H), 2.02 (s, 3H),2.05 (s, 3H), 2.35 (s, 3H), 3.00 (m, 1H), 3.09 (s, 3H), 3.3 (m, 1H),3.45 (s, 1H), 3.58 (d, J=6 Hz, 1H), 3.67 (d, J=10 Hz, 1H), 3.8-4.2 (m,4H), 4.72 (d, J=10 Hz, 1H), 5.13 (dd, J=12 and 7 Hz, 1H), 6.03 (dd, J=16and 7 Hz, 1H), 6.18 (d, J=12 Hz, 1H), 6.27 (d, J=10 Hz, 1H), 6.38 (m,1H), 6.44 (s, 1H), 7.10 (m, 1H), 7.37 (m, 4H), 8.21 (s, 1H), 8.88 (s,1H), 14.56 (s, 1H).

Preparation of RTI-9111-deoxy-11-imino-4-deoxy-3,4[2-spiro-[1-(3,3-dimethylbutanovl)-piperidin-4-yl]]-(1H)-imidazo-(2,5-dihydro)rifamycinS

Following the general procedure (A), the title compound was obtained asa pure solid. HRMS (ESI⁺): 910.4589 (M+Na)⁺; calculated for (M+Na)⁺:910.4579; RTI-91A, 1H-NMR (300 MHz, CDCl3): −0.07 (d, J=7 Hz, 3H), 0.61(d, J=7 Hz, 3H), 0.85 (d, J=7 Hz, 3H), 1.05 (m, 3H), 1.10 (s, 9H), 1.45(m, 1H), 1.6-1.85 (m, 4H), 1.88 (s, 3H), 2.02 (s, 3H), 2.05 (s, 3H),2.0-2.2 (m, 2H), 2.35 (s, 3H), 2.3-2.45 (m, 3H), 3.00 (m, 1H), 3.09 (s,3H), 3.33 (m, 1H), 3.47 (s, 0.4H), 3.52 (s, 0.6H), 3.55-3.70 (m, 3H),3.8-4.0 (m, 2H), 4.5 (m, 1H), 4.75 (m, 1H), 5.06 (m, 1H), 6.0 (m, 1H),6.17 (m, 1H), 6.29 (d, J=10 Hz, 1H), 6.4 (m, 1H), 8.27 (s, 1H), 8.63 (s,0.6H), 8.71 (s, 0.4H), 12.92 (s, 1H), 14.16 (s, 0.4H), 14.20 (s, 0.6H).

Preparation of RTI-9411-deoxy-11-imino-4-deoxy-3,4[2-spiro-[1-(n-pentanoyl)-piperidin-4-yl]]-(1H)-imidazo-(2,5-dihydro)rifamycinS

Following the general procedure (A), the title compound was obtained asa pure solid. HRMS (ESI⁺): 874.4644 (M+H)⁺; calculated for (M+H)⁺:874.4604; RTI-94A, 1H-NMR (300 MHz, CDCl3): −0.07 (d, J=7 Hz, 3H), 0.61(d, J=7 Hz, 3H), 0.84 (d, J=7 Hz, 3H), 0.97 (t, J=7 Hz, 3H), 1.04 (m,3H), 1.42 (m, 3H), 1.6-1.85 (m, 6H), 1.88 (s, 3H), 2.02 (s, 3H), 2.05(s, 3H), 1.9-2.2 (m, 2H), 2.35 (s, 3H), 2.3-2.45 (m, 3H), 3.00 (m, 1H),3.09 (s, 3H), 3.33 (m, 1H), 3.49 (s, 0.4H), 3.53 (s, 0.6H), 3.55-3.70(m, 3H), 3.8-4.0 (m, 2H), 4.5 (m, 1H), 4.72 (m, 1H), 5.06 (m, 1H), 6.0(m, 1H), 6.17 (m, 1H), 6.29 (d, J=10 Hz, 1H), 6.4 (m, 1H), 8.29 (s, 1H),8.63 (s, 0.6H), 8.70 (s, 0.4H), 12.92 (s, 1H), 14.17 (s, 0.4H), 14.20(s, 0.6H).

Preparation of RTI-9711-deoxy-11-imino-4-deoxy-3,4[2-spiro-[1-(2-methylpropanoyl)-piperidin-4-yl]]-(1H)-imidazo-(2,5-dihydro)rifamycinS

Following the general procedure (A), the title compound was obtained asa pure solid. HRMS (ESI⁺): 860.4482 (M+H)⁺; calculated for (M+H)⁺:860.4447. RTI-97A, 1H-NMR (300 MHz, CDCl3): −0.07 (d, J=7 Hz, 3H), 0.61(d, J=7 Hz, 3H), 0.84 (d, J=7 Hz, 3H), 1.04 (m, 3H), 1.20 (d, J=7 Hz,6H), 1.43 (m, 1H), 1.6-1.85 (m, 4H), 1.88 (s, 3H), 2.02 (s, 3H), 2.05(s, 3H), 2.0-2.2 (m, 2H), 2.35 (s, 3H), 2.40 (m, 1H), 2.89 (m, 1H), 3.01(m, 1H), 3.09 (s, 3H), 3.33 (m, 1H), 3.47 (s, 0.4H), 3.50 (s, 0.6H),3.55-3.70 (m, 3H), 3.8-4.1 (m, 2H), 4.5 (m, 1H), 4.72 (m, 1H), 5.06 (m,1H), 6.01 (dd, J=15 and 6 Hz, 1H), 6.18 (d, J=12 Hz, 1H), 6.29 (d, J=10Hz, 1H), 6.39 (m, 1H), 8.25 (s, 1H), 8.67 (s, 0.6H), 8.70 (s, 0.4H),12.93 (s, 1H), 14.16 (s, 0.4H), 14.19 (s, 0.6H).

Preparation of RTI-9811-deoxy-11-imino-4-deoxy-3,4[2-spiro-[1-(3-methylbutanoyl)-piperidin-4-yl]]-(1H)-imidazo-(2,5-dihydro)rifamycinS

Following the general procedure (A), the title compound was obtained asa pure solid. HRMS (ESI⁺): 874.4632 (M+H)⁺; calculated for (M+H)⁺:874.4604. RTI-98A, 1H-NMR (300 MHz, CDCl3): −0.07 (d, J=7 Hz, 3H), 0.61(d, J=7 Hz, 3H), 0.84 (d, J=7 Hz, 3H), 1.04 (m, 3H), 1.02 (d, J=7 Hz,6H), 1.43 (m, 1H), 1.6-1.85 (m, 4H), 1.88 (s, 3H), 2.02 (s, 3H), 2.05(s, 3H), 2.0-2.2 (m, 3H), 2.30 (m, 2H), 2.35 (s, 3H), 2.40 (m, 1H), 3.00(m, 1H), 3.09 (s, 3H), 3.33 (m, 1H), 3.47 (s, 0.4H), 3.50 (s, 0.6H),3.55-3.70 (m, 3H), 3.8-4.0 (m, 2H), 4.5 (m, 1H), 4.72 (m, 1H), 5.06 (m,1H), 6.01 (m, 1H), 6.17 (d, J=12 Hz, 0.6H), 6.18 (d, J=12 Hz, 0.4H),6.29 (d, J=10 Hz, 1H), 6.40 (m, 1H), 8.24 (s, 1H), 8.65 (s, 0.6H), 8.72(s, 0.4H), 12.92 (s, 1H), 14.16 (s, 0.4H), 14.19 (s, 0.6H).

Preparation of RTI-1014-deoxy-3,4[2-spiro-[1-(dimethylaminocarbonyl)-piperidin-4-yl]]-(1H)-imidazo-(2,5-dihydro)rifamycinS

Following the general procedure (B), the title compound was obtained asa pure solid. HRMS (ESI⁺): 884.4036 (M+Na)⁺; calculated for (M+Na)⁺:884.4058; RTI-101, 1H-NMR (300 MHz, CDCl3): −0.04 (d, J=7 Hz, 3H), 0.61(d, J=7 Hz, 3H), 0.84 (d, J=7 Hz, 3H), 1.04 (d, J=7 Hz, 3H), 1.44 (m,1H), 1.6 (m, 1H), 1.65-1.90 (m, 3H), 1.75 (s, 3H), 1.95-2.2 (m, 2H),2.01 (s, 3H), 2.04 (s, 3H), 2.35 (s, 3H), 2.40 (m, 1H), 2.90 (s, 6H),3.00 (m, 1H), 3.09 (s, 3H), 3.33 (m, 1H), 3.42 (s, 1H), 3.57 (d, J=6 Hz,1H), 3.6-3.8 (m, 5H), 4.72 (d, J=10 Hz, 1H), 5.14 (dd, J=12 and 7 Hz,1H), 6.00 (dd, J=16 and 7 Hz, 1H), 6.18 (d, J=12 Hz, 1H), 6.27 (d, J=10Hz, 1H), 6.37 (m, 1H), 8.19 (s, 1H), 8.96 (s, 1H), 14.62 (s, 1H).

Preparation of RTI-1024-deoxy-3,4[2-spiro-[1-(isobutylaminocarbonyl)-piperidin-4-yl]]-(1H)-imidazo-(2,5-dihydro)rifamycinS

Following the general procedure (B), the title compound was obtained asa pure solid. HRMS (ESI⁺): 912.4326 (M+Na)⁺; calculated for (M+Na)⁺:912.4371; RTI-102, 1H-NMR (300 MHz, CDCl3): −0.04 (d, J=7 Hz, 3H), 0.61(d, J=7 Hz, 3H), 0.84 (d, J=7 Hz, 3H), 0.95 (d, J=7 Hz, 6H), 1.04 (d,J=7 Hz, 3H), 1.44 (m, 1H), 1.6 (m, 1H), 1.65-1.90 (m, 4H), 1.75 (s, 3H),1.95-2.2 (m, 2H), 2.02 (s, 3H), 2.05 (s, 3H), 2.35 (s, 3H), 2.40 (m,1H), 3.00 (m, 1H), 3.09 (s, 3H), 3.12 (m, 2H), 3.33 (m, 1H), 3.45 (s,1H), 3.58 (d, J=6 Hz, 1H), 3.65 (d, J=10 Hz, 1H), 3.7-4.0 (m, 4H), 4.62(m, 1H), 4.73 (d, J=10 Hz, 1H), 5.13 (dd, J=12 and 7 Hz, 1H), 6.00 (dd,J=16 and 7 Hz, 1H), 6.18 (d, J=12 Hz, 1H), 6.27 (d, J=10 Hz, 1H), 6.38(m, 1H), 8.20 (s, 1H), 8.89 (s, 1H), 14.58 (s, 1H).

Preparation of RTI-1034-deoxy-3,4[2-spiro-[1-(isopropylaminocarbonyl)-piperidin-4-yl]]-(1H)-imidazo-(2,5-dihydro)rifamycinS

Following the general procedure (B), the title compound was obtained asa pure solid. HRMS (ESI⁺): 898.4194 (M+Na)⁺; calculated for (M+Na)⁺:898.4215; RTI-103, 1H-NMR (300 MHz, CDCl3): −0.04 (d, J=7 Hz, 3H), 0.61(d, J=7 Hz, 3H), 0.84 (d, J=7 Hz, 3H), 1.04 (d, J=7 Hz, 3H), 1.21 (d,J=7 Hz, 6H), 1.44 (m, 1H), 1.55 (m, 1H), 1.65-1.90 (m, 3H), 1.75 (s,3H), 2.0-2.15 (m, 2H), 2.02 (s, 3H), 2.05 (s, 3H), 2.35 (s, 3H), 2.40(m, 1H), 3.00 (m, 1H), 3.09 (s, 3H), 3.33 (m, 1H), 3.45 (s, 1H), 3.58(d, J=6 Hz, 1H), 3.66 (d, J=10 Hz, 1H), 3.7-4.0 (m, 4H), 4.03 (m, 1H),4.33 (d, J=7 Hz, 1H), 4.73 (d, J=10 Hz, 1H), 5.13 (dd, J=12 and 7 Hz,1H), 6.00 (dd, J=16 and 7 Hz, 1H), 6.18 (d, J=12 Hz, 1H), 6.27 (d, J=10Hz, 1H), 6.38 (m, 1H), 8.20 (s, 1H), 8.89 (s, 1H), 14.59 (s, 1H).

Preparation of RTI-1044-deoxy-3,4[2-spiro-[1-((1-methylpropyl)aminocarbonyl)-piperidin-4-yl]]-(1H)-imidazo-(2,5-dihydro)rifamycinS

Following the general procedure (B), the title compound was obtained asa pure solid. HRMS (ESI⁺): 912.4337 (M+Na)⁺; calculated for (M+Na)⁺:912.4371: RTI-104, 1H-NMR (300 MHz, CDCl3): −0.04 (d, J=7 Hz, 3H), 0.61(d, J=7 Hz, 3H), 0.84 (d, J=7 Hz, 3H), 0.95 (t, J=7 Hz, 3H), 1.04 (d,J=7 Hz, 3H), 1.18 (d, J=7 Hz, 3H), 1.4-1.6 (m, 4H), 1.65-1.85 (m, 3H),1.75 (s, 3H), 2.0-2.15 (m, 2H), 2.02 (s, 3H), 2.05 (s, 3H), 2.35 (s,3H), 2.40 (m, 1H), 3.00 (m, 1H), 3.09 (s, 3H), 3.33 (m, 1H), 3.45 (s,1H), 3.58 (d, J=6 Hz, 1H), 3.66 (d, J=10 Hz, 1H), 3.7-4.0 (m, 5H), 4.30(d, J=8 Hz, 1H), 4.73 (d, J=10 Hz, 1H), 5.13 (dd, J=12 and 7 Hz, 1H),6.00 (dd, J=16 and 7 Hz, 1H), 6.18 (d, J=12 Hz, 1H), 6.27 (d, J=10 Hz,1H), 6.38 (m, 1H), 8.20 (s, 1H), 8.89 (s, 1H), 14.59 (s, 1H).

Preparation of RTI-1054-deoxy-3,4[2-spiro-[1-(t-butylaminocarbonyl)-piperidin-4-yl]]-(1H)-imidazo-(2,5-dihydro)rifamycinS

Following the general procedure (B), the title compound was obtained asa pure solid. HRMS (ESI⁺): 912.4333 (M+Na); calculated for (M+Na)⁺:912.4371; RTI-105, 1H-NMR (300 MHz, CDCl3): −0.05 (d, J=7 Hz, 3H), 0.61(d, J=7 Hz, 3H), 0.84 (d, J=7 Hz, 3H), 1.04 (d, J=7 Hz, 3H), 1.40 (s,9H), 1.4-1.6 (m, 2H), 1.7-1.85 (m, 3H), 1.75 (s, 3H), 2.0-2.15 (m, 2H),2.01 (s, 3H), 2.05 (s, 3H), 2.35 (s, 3H), 2.40 (m, 1H), 3.00 (m, 1H),3.09 (s, 3H), 3.33 (m, 1H), 3.46 (s, 1H), 3.59 (d, J=6 Hz, 1H), 3.66 (d,J=10 Hz, 1H), 3.7-4.0 (m, 4H), 4.43 (s, 1H), 4.73 (d, J=10 Hz, 1H), 5.13(dd, J=12 and 7 Hz, 1H), 6.00 (dd, J=16 and 7 Hz, 1H), 6.18 (d, J=12 Hz,1H), 6.27 (d, J=10 Hz, 1H), 6.38 (m, 1H), 8.22 (s, 1H), 8.87 (s, 1H),14.60 (s, 1H).

Preparation of RTI-175 11-deoxy-11-hydroxy-4-deoxy-34[2-spiro-[1-(isobutyloxycarbonyl)-piperidin-4-yl]]-(1H)-imidazo-(2,5-dihydro)rifamycinS

Following the general procedure (C), the title compound was obtained asa pure solid. HRMS (ESI⁺): 915.4334 (M+Na)⁺; calculated for (M+Na)⁺:915.4368; RTI-175, 1H-NMR (300 MHz, CDCl3): 0.05 (d, J=7 Hz, 3H), 0.63(d, J=7 Hz, 3H), 0.85 (d, J=7 Hz, 3H), 0.96 (d, J=7 Hz, 6H), 1.04 (d,J=7 Hz, 3H), 1.40-1.60 (m, 2H), 1.7-2.1 (m, 6H), 1.93 (s, 3H), 2.05 (s,3H), 2.07 (s, 3H), 2.24 (s, 3H), 2.40 (m, 1H), 3.00 (m, 1H), 3.07 (s,3H), 3.48 (m, 1H), 3.68 (s, 1H), 3.5-3.8 (m, 2H), 3.86 (d, J=6 Hz, 2H),3.85-4.1 (m, 4H), 4.95 (dd, J=12 and 4 Hz, 1H), 5.05 (d, J=10 Hz, 1H),5.54 (s, 1H), 5.99 (d, J=12 Hz, 1H), 6.16 (dd, J=16 and 6 Hz, 1H), 6.27(d, J=10 Hz, 1H), 6.44 (dd, J=16 and 10 Hz, 1H), 6.72 (s, 1H), 8.07 (s,1H), 8.22 (bs, 1H), 13.61 (s, 1H).

Preparation of RTI-17611-deoxy-11-amino-4-deoxy-3,4[2-spiro-[1-(isobutyloxycarbonyl)-piperidin-4-yl]]-(1H)-imidazo-(2,5-dihydro)rifamycinS

Following the general procedure (C), the title compound was obtained asa pure solid. HRMS (ESI⁺): 892.4689 (M+H)⁺; calculated for (M+H)⁺:892.4710; RTI-176 (RTI2-63B, 1H-NMR (300 MHz) (CDCl3): −0.05 (d, J=7 Hz,3H), 0.64 (d, J=7 Hz, 3H), 0.85 (d, J=7 Hz, 3H), 0.96 (d, J=7 Hz, 6H),1.04 (d, J=7 Hz, 3H), 1.40-1.70 (m, 2H), 1.7-1.9 (m, 4H), 1.9-2.1 (m,2H), 1.94 (s, 3H), 2.05 (s, 3H), 2.08 (s, 3H), 2.24 (s, 3H), 2.40 (m,1H), 2.6-2.8 (br, 2H), 3.03 (m, 1H), 3.07 (s, 3H), 3.52 (m, 1H), 3.67(s, 1H), 3.6-3.7 (m, 2H), 3.80 (d, J=10 Hz, 1H), 3.91 (d, J=6 Hz, 2H),3.85-4.1 (m, 2H), 4.11 (d, J=4 Hz, 1H), 4.77 (s, 1H), 4.87 (dd, J=12 and4 Hz, 1H), 5.09 (d, J=10 Hz, 1H), 5.98 (d, J=12 Hz, 1H), 6.18 (dd, J=16and 6 Hz, 1H), 6.25 (d, J=10 Hz, 1H), 6.44 (dd, J=16 and 11 Hz, 1H),8.19 (s, 1H), 8.24 (bs, 1H), 13.93 (s, 1H).

Preparation of RTI-18111-deoxy-11-amino-4-deoxy-3,4[2-spiro-[1-(2-methylpropyl)-piperidin-4-yl]]-(1H)-imidazo-(2,5-dihydro)rifamycinS

Following the general procedure (C), the title compound was obtained asa pure solid. HRMS (ESI⁺): 848.4777 (M+H)⁺; calculated for (M+H)⁺:848.4811; RTI-181, 1H-NMR (300 MHz, CDCl3): −0.05 (d, J=7 Hz, 3H), 0.63(d, J=7 Hz, 3H), 0.85 (d, J=7 Hz, 3H), 0.92 (d, J=6 Hz, 6H), 1.04 (d,J=7 Hz, 3H), 1.40-1.50 (m, 1H), 1.7-2.1 (m, 9H), 1.94 (s, 3H), 2.05 (s,3H), 2.07 (s, 3H), 2.23 (s, 3H), 2.24 (m, 2H), 2.40 (m, 1H), 2.6-2.8 (m,4H), 3.03 (m, 1H), 3.07 (s, 3H), 3.50 (m, 1H), 3.68 (s, 1H), 3.80 (d,J=10 Hz, 1H), 4.11 (d, J=4 Hz, 1H), 4.76 (s, 1H), 4.87 (dd, J=12 and 4Hz, 1H), 5.09 (d, J=10 Hz, 1H), 5.98 (d, J=12 Hz, 1H), 6.18 (dd, J=16and 6 Hz, 1H), 6.25 (d, J=10 Hz, 1H), 6.44 (dd, J=16 and 11 Hz, 1H),8.27 (s, 1H), 8.32 (s, 1H), 14.03 (s, 1H).

Preparation of RTI-18211-deoxy-11-imino-4-deoxy-3,4[2-spiro-[1-(isobutylaminocarbonyl)-piperidin-4-yl]]-(1H)-imidazo-(2,5-dihydro)rifamycinS

Following the general procedure (A), the title compound was obtained asa pure solid. HRMS (ESI⁺): 889.4678 (M+H)⁺; calculated for (M+H)⁺:889.4713; RTI-182, 1H-NMR (300 MHz, CDCl3): −0.08 (d, J=7 Hz, 3H), 0.61(d, J=7 Hz, 3H), 0.84 (d, J=7 Hz, 3H), 0.96 (d, J=7 Hz, 6H), 1.04 (d,J=7 Hz, 3H), 1.4 (m, 1H), 1.65 (m, 1H), 1.7-1.85 (m, 4H), 1.88 (s, 3H),1.95-2.15 (m, 2H), 2.02 (s, 3H), 2.05 (s, 3H), 2.34 (s, 3H), 2.40 (m,1H), 3.00 (m, 1H), 3.09 (s, 3H), 3.12 (m, 2H), 3.33 (m, 1H), 3.50 (s,1H), 3.62 (d, J=5 Hz, 1H), 3.67 (d, J=9 Hz, 1H), 3.6-3.7 (m, 2H),3.8-4.0 (m, 2H), 4.62 (t, J=5 Hz, 1H), 4.72 (d, J=10 Hz, 1H), 5.06 (dd,J=12 and 7 Hz, 1H), 6.02 (dd, J=15 and 7 Hz, 1H), 6.16 (d, J=12 Hz, 1H),6.29 (d, J=10 Hz, 1H), 6.38 (m, 1H), 8.27 (s, 1H), 8.67 (s, 1H), 12.92(s, 1H), 14.58 (s, 1H).

Preparation of RTI-18311-deoxy-11-amino-4-deoxy-3,4[2-spiro-[1-(isobutylaminocarbonyl)-piperidin-4-yl]]-(1H)-imidazo-(2,5-dihydro)rifamycinS

Following the general procedure (C), the title compound was obtained asa pure solid. HRMS (ESI⁺): 891.4843 (M+H)⁺; calculated for (M+H)⁺:891.4870; RTI-183, 1H-NMR (300 MHz, CDCl3): −0.05 (d, J=7 Hz, 3H), 0.64(d, J=7 Hz, 3H), 0.85 (d, J=7 Hz, 3H), 0.94 (d, J=7 Hz, 6H), 1.04 (d,J=7 Hz, 3H), 1.48 (m, 1H), 1.7-1.89 (m, 8H), 1.94 (s, 3H), 2.01 (m, 1H),2.04 (s, 3H), 2.08 (s, 3H), 2.24 (s, 3H), 2.40 (m, 1H), 3.03 (m, 1H),3.07 (s, 3H), 3.09 (m, 2H), 3.52 (m, 1H), 3.55-3.75 (m, 3H), 3.75 (s,1H), 3.81 (d, J=10 Hz, 1H), 3.85-4.0 (m, 1H), 4.13 (d, J=4 Hz, 1H), 4.62(t, J=5 Hz, 1H), 4.77 (s, 1H), 4.88 (dd, J=12 and 4 Hz, 1H), 5.09 (d,J=10 Hz, 1H), 5.98 (d, J=12 Hz, 1H), 6.18 (dd, J=16 and 6 Hz, 1H), 6.26(d, J=10 Hz, 1H), 6.44 (dd, J=16 and 11 Hz, 1H), 8.20 (s, 1H), 8.35 (s,1H), 13.94 (s, 1H).

Preparation of RTI-754-deoxy-3,4[2-spiro-[1-(t-butyloxycarbonvl)-piperidin-4-yl]]-(1H)-imidazo-(2,5-dihydro)rifamycinS

Following the general procedure (B), the title compound was obtained asa pure solid. HRMS (ESI⁺): 913.4267 (M+Na)⁺; calculated for (M+Na)⁺:913.4211; RTI-75A, 1H-NMR (300 MHz, CDCl3): −0.04 (d, J=7 Hz, 3H), 0.61(d, J=7 Hz, 3H), 0.84 (d, J=7 Hz, 3H), 1.04 (d, J=7 Hz, 3H), 1.40-1.60(m, 2H), 1.51 (s, 9H), 1.7-1.85 (m, 3H), 1.75 (s, 3H), 1.9-2.1 (m, 2H),2.02 (s, 3H), 2.05 (s, 3H), 2.35 (s, 3H), 2.40 (m, 1H), 3.00 (m, 1H),3.09 (s, 3H), 3.33 (m, 1H), 3.43 (s, 1H), 3.57 (d, J=5 Hz, 1H), 3.67 (d,J=10 Hz, 1H), 3.6-3.8 (br, 2H), 3.9-4.1 (br, 2H), 4.72 (d, J=10 Hz, 1H),5.13 (dd, J=12 and 7 Hz, 1H), 6.02 (dd, J=16 and 7 Hz, 1H), 6.18 (d,J=12 Hz, 1H), 6.28 (d, J=10 Hz, 1H), 6.40 (dd, J=16 and 10 Hz, 1H), 8.19(s, 1H), 8.93 (bs, 1H), 14.59 (s, 1H).

Preparation of RTI-764-deoxy-3,4[2-spiro-[1-(ethyloxycarbonyl)-piperidin-4-yl]]-(1H)-imidazo-(2,5-dihydro)rifamycinS

Following the general procedure (B), the title compound was obtained asa pure solid. HRMS (ESI⁺): 885.3945 (M+Na)⁺; calculated for (M+Na)⁺885.3898; RTI-76A, 1H-NMR (300 MHz, CDCl3): −0.04 (d, J=7 Hz, 3H), 0.61(d, J=7 Hz, 3H), 0.84 (d, J=7 Hz, 3H), 1.04 (d, J=7 Hz, 3H), 1.30 (t,J=7 Hz, 3H), 1.40-1.60 (m, 2H), 1.7-1.85 (m, 3H), 1.75 (s, 3H), 1.9-2.1(m, 2H), 2.02 (s, 3H), 2.05 (s, 3H), 2.35 (s, 3H), 2.40 (m, 1H), 3.00(m, 1H), 3.09 (s, 3H), 3.33 (m, 1H), 3.44 (s, 1H), 3.57 (d, J=5 Hz, 1H),3.66 (d, J=10 Hz, 1H), 3.7-3.9 (br, 2H), 4.0-4.2 (br, 2H), 4.21 (q, J=7Hz, 2H), 4.72 (d, J=10 Hz, 1H), 5.13 (dd, J=12 and 7 Hz, 1H), 6.00 (dd,J=16 and 7 Hz, 1H), 6.18 (d, J=12 Hz, 1H), 6.27 (d, J=10 Hz, 1H), 6.40(dd, J=16 and 10 Hz, 1H), 8.20 (s, 1H), 8.92 (bs, 1H), 14.58 (s, 1H).

Preparation of RTI-784-deoxi-3,4[2-spiro-[1-(n-propyloxcarbonyl)-piperidin-4-ylJJ]]-(1H)-imidazo-(2,5-dihydro)rifamycinS

Following the general procedure (B), the title compound was obtained asa pure solid. HRMS (ESI⁺): 899.3989 (M+Na)⁺; calculated for (M+Na)⁺899.4054; RTI-78A, 1H-NMR (300 MHz, CDCl3): −0.04 (d, J=7 Hz, 3H), 0.61(d, J=7 Hz, 3H), 0.84 (d, J=7 Hz, 3H), 0.99 (t, J=7 Hz, 3H), 1.04 (d,J=7 Hz, 3H), 1.40-1.60 (m, 2H), 1.69 (m, 2H), 1.7-1.85 (m, 3H), 1.75 (s,3H), 1.95-2.1 (m, 2H), 2.02 (s, 3H), 2.05 (s, 3H), 2.35 (s, 3H), 2.40(m, 1H), 3.00 (m, 1H), 3.09 (s, 3H), 3.33 (m, 1H), 3.42 (s, 1H), 3.56(d, J=5 Hz, 1H), 3.66 (d, J=10 Hz, 1H), 3.7-3.9 (br, 2H), 4.0-4.2 (br,2H), 4.11 (t, J=7 Hz, 2H), 4.72 (d, J=10 Hz, 1H), 5.13 (dd, J=12 and 7Hz, 1H), 6.00 (dd, J=16 and 7 Hz, 1H), 6.18 (d, J=12 Hz, 1H), 6.27 (d,J=10 Hz, 1H), 6.40 (dd, J=16 and 10 Hz, 1H), 8.17 (s, 1H), 8.92 (bs,1H), 14.57 (s, 1H).

Preparation of RTI-794-deoxy-3,4[2-spiro-[1-(isobutyloxycarbonyl)-piperidin-4-yl]]-(1H)-imidazo-(2,5-dihydro)rifamycinS

Following the general procedure (B), the title compound was obtained asa pure solid. HRMS (ESI⁺): 913.4163 (M+Na)⁺; calculated for (M+Na)⁺913.4211; RTI-79A, 1H-NMR (300 MHz, CDCl3): −0.03 (d, J=7 Hz, 3H), 0.61(d, J=7 Hz, 3H), 0.84 (d, J=7 Hz, 3H), 0.97 (d, J=7 Hz, 6H), 1.04 (d,J=7 Hz, 3H), 1.40-1.60 (m, 2H), 1.7-1.85 (m, 3H), 1.75 (s, 3H), 1.9-2.1(m, 3H), 2.02 (s, 3H), 2.05 (s, 3H), 2.35 (s, 3H), 2.40 (m, 1H), 3.00(m, 1H), 3.09 (s, 3H), 3.33 (m, 1H), 3.42 (s, 1H), 3.56 (d, J=5 Hz, 1H),3.66 (d, J=10 Hz, 1H), 3.7-3.9 (br, 2H), 3.93 (d, J=6 Hz, 2H), 4.0-4.2(br, 2H), 4.72 (d, J=10 Hz, 1H), 5.13 (dd, J=12 and 7 Hz, 1H), 6.00 (dd,J=16 and 7 Hz, 1H), 6.19 (d, J=12 Hz, 1H), 6.27 (d, J=10 Hz, 1H), 6.39(dd, J=16 and 10 Hz, 1H), 8.17 (s, 1H), 8.93 (bs, 1H), 14.57 (s, 1H).

Preparation of RTI-804-deoxy-3,4[2-spiro-[1-(beRTIyloxycarbonyl)-piperidin-4-yl]]-(1H)-imidazo-(2,5-dihydro)rifamycinS

Following the general procedure (B), the title compound was obtained asa pure solid. HRMS (ESI⁺): 947.3987 (M+Na)⁺; calculated for (M+Na)⁺947.4054; RTI-80A, 1H-NMR (300 MHz, CDCl3): −0.04 (d, J=7 Hz, 3H), 0.61(d, J=7 Hz, 3H), 0.84 (d, J=7 Hz, 3H), 1.04 (d, J=7 Hz, 3H), 1.40-1.60(m, 2H), 1.7-1.85 (m, 3H), 1.74 (s, 3H), 1.9-2.1 (m, 2H), 2.01 (s, 3H),2.04 (s, 3H), 2.35 (s, 3H), 2.40 (m, 1H), 3.00 (m, 1H), 3.09 (s, 3H),3.33 (br, 1H), 3.42 (br, 1H), 3.56 (d, J=5 Hz, 1H), 3.66 (d, J=10 Hz,1H), 3.7-3.9 (br, 2H), 4.0-4.2 (br, 2H), 4.72 (d, J=10 Hz, 1H), 5.13(dd, J=12 and 7 Hz, 1H), 5.20 (m, 2H), 6.00 (dd, J=16 and 7 Hz, 1H),6.18 (d, J=12 Hz, 1H), 6.27 (d, J=10 Hz, 1H), 6.39 (dd, J=16 and 10 Hz,1H), 7.39 (m, 5H), 8.16 (s, 1H), 8.93 (bs, 1H), 14.57 (s, 1H).

Preparation of RTI-17411-deoxy-11-hydroxy-4-deoxy-3,4[2-spiro-[1-(2-methylpropvl)-piperidin-4-yl]]-(1H)-imidazo-(2,5-dihydro)rifamycinS

Following the general procedure (C), the title compound was obtained asa pure solid. HRMS (ESI⁺): 871.4433 (M+Na)⁺; calculated for (M+Na)⁺871.4470.

Preparation of RTI-19711-deoxy-11-hydroxyimino-4-deoxy-3,4[2-spiro-[1-(isobutyloxycarbonyl)-piperidin-4-yl]]-(1H)-imidazo-(2,5-dihydro)rifamycinS

Following the general procedure (D), the title compound was obtained asa solid. HRMS (ESI⁺): 906.4535 (M+H)⁺; calculated for (M+H)⁺ 906.4535;RTI-197, 1H-NMR (300 MHz, CDCl3): −0.03 (d, J=7 Hz, 3H), 0.62 (d, J=7Hz, 3H), 0.84 (d, J=7 Hz, 3H), 0.97 (d, J=7 Hz, 6H), 1.04 (d, J=7 Hz,3H), 1.35-1.40 (m, 1H), 1.7-1.8 (m, 1H), 1.85-2.1 (m, 6H), 2.00 (s, 3H),2.04 (s, 3H), 2.13 (s, 3H), 2.33 (s, 3H), 2.40 (m, 1H), 3.00 (m, 1H),3.10 (s, 3H), 3.34 (m, 1H), 3.42-3.50 (m, 2H), 3.67 (d, J=10 Hz, 1H),3.8-3.9 (m, 4H), 3.93 (d, J=6 Hz, 2H), 4.60 (d, J=10 Hz, 1H), 5.23 (dd,J=12 and 8 Hz, 1H), 5.98 (dd, J=15 and 6 Hz, 1H), 6.30 (d, J=12 Hz, 2H),6.40 (dd, J=16 and 10 Hz, 1H), 8.35 (s, 1H), 8.92 (bs, 1H), 14.13 (s,1H).

Preparation of RTI-21711-deoxy-11-hydroxyimino-4-deoxy-3,4[2-spiro-[1-(isobutylaminocarbonyl)-piperidin-4-yl]]-(1H)-imidazo-(2,5-dihydro)rifamycinS

Following the general procedure (D), the title compound was obtained asa solid. HRMS (ESI⁺): 905.4695 (M+H)⁺: calculated for (M+H)⁺ 905.4662;RTI-217, 1H-NMR (300 MHz, CDCl3): −0.03 (d, J=7 Hz, 3H), 0.62 (d, J=7Hz, 3H), 0.84 (d, J=7 Hz, 3H), 0.95 (d, J=7 Hz, 6H), 1.04 (d, J=7 Hz,3H), 1.35-1.40 (m, 1H), 1.7-1.8 (m, 1H), 1.85-2.1 (m, 6H), 2.00 (s, 3H),2.04 (s, 3H), 2.13 (s, 3H), 2.33 (s, 3H), 2.40 (m, 1H), 3.00 (m, 1H),3.10 (s, 3H), 3.08-3.14 (m, 2H), 3.34 (m, 1H), 3.45 (s, 1H), 3.47 (d,J=6 Hz, 1H), 3.65-3.8 (m, 5H), 4.60 (m, 2H), 5.23 (dd, J=12 and 8 Hz,1H), 5.98 (dd, J=16 and 7 Hz, 1H), 6.30 (d, J=12 Hz, 2H), 6.40 (dd, J=16and 10 Hz, 1H), 8.34 (s, 1H), 8.89 (s, 1H), 14.14 (s, 1H).

Example 13 Preparation of a Rifabutin Derivative Modifed on AlterativeSites

Biotin-glycine-substituted rifabutin derivative RTI-173 contains asubstitution at the 21-hydroxy site, yet has a similar activity asrifabutin on G3 cells when combined with doxorubicin, suggesting thatthis site may be modified without affecting drug-sensitization or cancerinhibition effects. Biotin-glycine-linked rifabutin derivative (RTI-173)has the following formula:

RTI-173 was prepared by the following method:

A solution of Glycine-rifabutin (240 mg, 0.27 mmole) in DMF (2 ml) wasadded to a solution of biotin (65 mg, 0.27 mmol), DMAP (33 mg, 0.27mmol) and EDCI (52 mg, 0.27 mmole) in DMF (3 ml) at room temperature.The reaction mixture stirred at room temperature overnight and dilutedwith DCM (40 ml) and washed with water and brine. The organic phase wasdried over anhydrous sodium sulphate, filtered and concentrated undervacuum. The residue was purified by silica gel column chromatographywith methanol in DCM as eluent to give 108 mg of the product as purplesolid. HRMS (ESI⁺): 1152.5538 (M+Na)⁺; calculated for (M+Na)⁺ 1152.5304.

Example 14 Example Rifabutin and Rifabutin Derivative Compositions andMethods of Administration to a Chemotherapeutic-Resistant Cancer Patient

Rifamycin and rifamycin derivatives, such as rifabutin and rifabutinderivative compositions may be prepared as described herein.Compositions formulated in the same ways as rifabutin, rifamycin, orrelated drugs typically currently formulated may be useful foradministration to cancer patients. These compositions may containrifamycin, a rifamycin derivative, rifabutin, or a rifabutin derivative,such as the RTI-79 derivative described herein.

In particular, compositions may be formulated in tablets or capsules fororal use. These tablets or capsules may be extended release tablets orcapsules to provide a more stable and continuous supply of the rifamycinor rifamycin derivative to the cancer cells in the patient. Tablets orcapsules may contain at least 10 mg, at least 50 mg, at least 100 mg, atleast 150 mg, or at least 200 mg of rifamycin or rifamycin derivative.Combination tablets or capsules with other drugs, such aschemotherapeutic drugs or other drugs commonly administered withchemotherapy may be prepared, particularly if the recommended dosingschedule for those drugs is similar to that of the rifamycin orrifamycin derivative. For example, the rifamycin or rifamycin derivativemay be combined with the prednisone portion of CHOP therapy or anothersteroid or other drug that is intended to be administered daily.

Compositions may also be formulated for intravenous injection as well.In general, the amount of rifamycin or rifamycin derivative, such asrifabutin or a rifabutin derivative, may be lower in a dose formulatedfor intravenous injection than in a dose formulated for oraladministration because intravenous injection avoids the need forabsorption through the intestines. Injectable doses of rifamycin orrifamycin derivative, including rifabutin or a rifabutin derivative, maybe provided in multi-use containers or in single-use containers. Thesecontainers may be compatible for use with standard intravenous needlesand syringes as well as intravenous drip systems and more complexchemotherapeutic administration systems. Single-use containers maycontain the entire amount of rifamycin or rifamycin derivativeadministered with a round a chemotherapy to avoid the need for multipleinjections of the drug. Alternatively, they may contain amountsappropriate for daily doses. Single-use containers may contain at least1 mg, at least 5 mg, at least 10 mg, at least 50 mg, at least 100 mg, orat least 150 mg of rifamycin or rifamycin derivative. Multi-usecontainers may be designed to allow administration of these same amountsof rifamycin or rifamycin derivative. Injectable compositions mayfurther contain other injectable chemotherapeutic drugs or other drugscommonly administered with chemotherapy. In one specific example,injectable compostions may contain doxorubicin or a similarchemotherapeutic in a liposome. In such compositions, the rifamycin orrifamycin derivate may also be in the liposome. In general, due toimprovements in delivery via liposomes, if the rifamycin or rifamycinderivative is contained in a liposome, the total amount in the dose maybe less than if the rifamycin or rifamycin derivative is injectable, butnot in a liposome.

Rifamycin and rifamycin derivatives, such as rifabutin and rifabutinderivatives may be administered to patients with cancer in the form ofany compositions described in this example or elsewhere herein or anyany other form. The patients with cancer may have a cancer that isresistant to one or more chemotherapeutics, may be at risk fordeveloping cancer resistant to one or more chemotherapeutics, maybenefit from administration of reduced amounts of one or morechemotherapeutics, or may benefit from the administration of aparticular chemotherapeutic to which rifamycin or a rifamycin derivativesensitizes the patent's cancer cells.

In one example, the rifamycin or rifamycin derivates may be administeredorally to patients with cancer. In particular, they may be administeredin the form of tablets or capsules. The rifamycin or rifamycinderivative may be administered such that the patient receives at least50 mg/adult human/week, at least 100 mg/adult human/week, at least 150mg/adult human/week, or at least 300 mg/adult human/week. Amounts may bereduced for children. For example, a child under age 5 might receive onequarter or less of an adult human dose. A child age 5 to age 10 mayreceive one half to one quarter the adult human dose. A child age 10 orover over may receive three quarters to one half the adult human dose.In another embodiment, the rifamycin or rifamycin derivative may beadministered such that the patient receives at least 0.5 mg/kg/week, atleast 1 mg/kg/week, at least 2 mg/kg/week, at least 5 mg/kg/week, atleast 10 mg/kg/week, at least 20 mg/kg/week, at least 30 mg/kg/week, atleast 50 mg/kg/week or at least 100 mg/kg/week.

Rifamycin or a rifamycin derivative administered orally in this fashionmay be administered weekly, daily, or multiple times per day. The dosingschedule may be adjusted so as to maintain minimal blood concentrationsfor a period of time, particularly with extended release formulations.Alternatively, maintenance of minimal blood concentrations may not benecessary for some methods of treatment and dosing may instead bedesigned to achieve a total blood concentration for a shorter period oftime, such as for four hours or less. Although amounts are expressed asweekly totals, it will be understood that the compositions do not haveto be administered for a full week. For example, a patient may receive asingle dose in connection with a chemotherapeutic treatment and may notreceive a further dose until much later, with another chemotherapeutictreatment, or not at all. Furthermore, it is possible to administer theweekly total through various combinations of doses on various days. Forexample, it may be possible to administer doses only every other day orevery few days. Doses also need not be the same each day. For example, apatient may receive doses that increase or decrease over time,particularly due to the schedule for administration ofchemotherapeutics. In one example, the patient may be provided with apack of varying-dose tablets or capsules labeled by day (e.g. Day 1, Day2, etc.), by portions of the day (e.g. Day 1 morning. Day 1 evening,etc.), or by week (e.g. Week 1, Week 2, etc.) and instructed to begintaking the tablets or capsules at a specified time dictated by theschedule for administration of a chemotherapeutic.

In general, the rifamycin or rifamycin derivative may be administered inconnection with administration of a chemotherapeutic. In one example, itmay be administered at least weekly or at least daily the entire timethe patient is receiving a course of a chemotherapeutic, such as forseveral months. In another example it may be administered only tocoincide with administration of a chemotherapetic, such as for one dayto one week each month coinciding with a once monthy chemotherapeuticadministration.

In one specific example, the rifamycin or rifamycin derivative may berifabutin or RTI-79 administered orally in one to three doses ofrifabutin or RTI-79 in 100 mg to 300 mg amounts over a period of up to48 hours beginning within 24 hours before or after the administration ofa chemotherapeutic, such as DOXIL®. A single oral dose of 300 mgrifabutin causes a mean (±SD) peak plasma concentration (Cmax) of 375(±267) ng/mL (range 141 to 1033 ng/mL). The plasma elimination ofrifabutin is biphasic with an initial half-life of approximately 4hours, followed by a mean terminal half-life of 45 (±17) hours (range 16to 69 hours). The rifabutin derivative RTI-79 is expected to presentsimilar results. Accordingly, appropriate dosages for variations of thisexample using intravenously injected rifabutin or RTI-79 rather thanorally administered forms may be calculated.

In an alternative embodiment, rifamycin or a rifamycin derivative, suchas rifabutin or RTI-79, may be administered in a method that matches thepharmokinetics of the rifamycin or rifamycin derivative to that of thechemotherapeutic also administered to the patient. For example, maximaldoxorubicin tissue absorption occurs 48 hours after administration.Maximal RTI-79 plasma concentration is reached within 3 hours ofadministration. Accordingly, administerting RTI-79 orally 24 and 48hours after intravenous doxorubicin administration may maximizeefficacy.

In another alternative embodiment, rifamycin or a rifamycin derivative,such as rifabutin or RTI-79, may be administered in amounts similar tothose described herein after the cessation of chemotherapy to reduce orprevent metastasis.

Although only exemplary embodiments of the invention are specificallydescribed above, it will be appreciated that modifications andvariations of these examples are possible without departing from thespirit and intended scope of the invention. For example, variousspecific formulations including components not listed herein andspecific methods of administering such formulations may be developedusing the ordinary skill in the art. Numeric amounts expressed hereinwill be understood by one of ordinary skill in the art to includeamounts that are approximately or about those expressed. Furthermore,the term “or” as used herein is not intended to express exclusiveoptions (either/or) unless the context specifically indicates thatexclusivity is required; rather “or” is intended to be inclusive(and/or).

The invention claimed is:
 1. A method of sensitizing a cancer cell to adrug comprising administering rifamycin or rifabutin to the cancer cellin an amount and for a time sufficient to sensitize the cancer cell tothe drug.
 2. The method of claim 1, further comprising administeringrifamycin or rifabutin to the cancer cell before the drug to which thecancer cell is sensitized.
 3. The method of claim 1, further comprisingadministering rifamycin or rifabutin to the cancer cell concurrentlywith the drug to which the cancer cell is sensitized.
 4. The method ofclaim 1, further comprising administering rifamycin or rifabutin to thecancer cell after the drug to which the cancer cell is sensitized. 5.The method of claim 1, further comprising administering the rifamycin orrifabutin to the cancer cell a second or greater time.
 6. The method ofclaim 1, wherein the cancer cell is a carcinoma, a sarcoma, as leukemia,a lymphoma, or a glioma.
 7. The method of claim 1, wherein the cancercell is a metastatic cancer cell.
 8. A method of inhibiting a cancercell with a drug comprising: administering rifamycin or rifabutin to thecancer cell in an amount and for a time sufficient to sensitize thecancer cell to the drug; and administering the drug to the cancer cellin an amount and for a time sufficient to inhibit the cancer cell,wherein the amount or time are less than that required to achieve thesame inhibition in the absence of rifamycin or rifabutin.
 9. The methodof claim 8, further comprising administering the rifamycin or rifabutinconcurrently with the drug.
 10. The method of claim 8, furthercomprising administering the rifamycin or rifabutin before administeringthe drug.
 11. The method of claim 8, further comprising administeringthe rifamycin or rifabutin after administering the drug.
 12. The methodof claim 8, further comprising administering rifamycin or rifabutin tothe cancer cell a second or greater time.
 13. The method of claim 8,wherein the drug is a chemotherapeutic drug.
 14. The method of claim 8,wherein the cancer cell is a carcinoma, a sarcoma, as leukemia, alymphoma, or a glioma.
 15. The method of claim 8, wherein the cancercell is a metastatic cancer cell.
 16. A method of increasingsusceptibility of a cancer cell to a drug comprising: administeringrifamycin or rifabutin to a cancer cell in an amount and for a timesufficient to increase the amount of the drug in the cancer cell ascompared to the amount of the drug that would be present in the absenceof the rifamycin rifabutin; and administering the drug to the cancercell in an amount and for a time sufficient to inhibit the cancer cell.17. The method of claim 16, wherein the drug comprises achemotherapeutic drug.
 18. The method of claim 16, wherein the cancercell comprises a cancer cell resistant to the drug.
 19. The method ofclaim 16, further comprising administering the rifamycin or rifabutinconcurrently with the drug.
 20. The method of claim 16, furthercomprising administering the rifamycin or rifabutin before administeringthe drug.
 21. The method of claim 16, further comprising administeringthe rifamycin or rifabutin after administering the drug.
 22. The methodof claim 16, further comprising administering rifamycin or rifabutin tothe cancer cell a second or greater time.
 23. The method of claim 16,wherein the drug is a chemotherapeutic drug.
 24. The method of claim 16,wherein the cancer cell is a carcinoma, a sarcoma, as leukemia, alymphoma, or a glioma.
 25. The method of claim 16, wherein the cancercell is a metastatic cancer cell.